LOCUS       sequence-448390-        8052 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression and inducible recruitment of protein in mammalian 
            cell culture.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8052)
  AUTHORS   Adriaenssens E, Schaar S, Cook ASI, Stuke JFM, Sawa-Makarska J, 
            Nguyen TN, Ren X, Schuschnig M, Romanov J, Khuu G, Uoselis L, 
            Lazarou M, Hummer G, Hurley JH, Martens S
  TITLE     Reconstitution of BNIP3/NIX-mitophagy initiation reveals 
            hierarchical flexibility of the autophagy machinery.
  JOURNAL   Nat Cell Biol. 2025 Jul 25. doi: 10.1038/s41556-025-01712-y.
  PUBMED    40715440
REFERENCE   2  (bases 1 to 8052)
  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: "Nat Cell 
            Biol. 2025 Jul 25. doi: 10.1038/s41556-025-01712-y."
COMMENT     Sequence Label: pHAGE-FKBP-mEGFP-WIPI2 IDR (364-425aa)
FEATURES             Location/Qualifiers
     source          1..8052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2062..2179
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2231..2534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2535..2738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2689..2709
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2735..2759
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2821..2830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2827..2829,2830..3150)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             3157..3477
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /note="synthetic gene with alternative codons"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3157..3477
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             join(3496..3498,3499..4209)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3538..3559)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3799..3818)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4146..4167
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4442..5030
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4495..4515)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4913..4924)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5105..5338
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5457..5475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5543..5647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5648..5716,5717..6508)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5866..5885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6679..7267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7168..7187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7421..7438
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7678..7699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7714..7719,7720..7737,7738..7744)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7752..7768
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7757..7779
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7776..7792
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7776..7792
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7842..7861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttaattcaca
     2041 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2101 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2161 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggactag
     2221 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2281 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2341 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2401 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2461 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2521 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     2581 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     2641 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     2701 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     2761 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccggcgg ccgcggtacc
     2821 gccaccatgg gagtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag
     2881 cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaatttgat
     2941 tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga
     3001 ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct
     3061 ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc
     3121 gtcttcgatg tggagcttct aaaactggaa actagaggcg tgcaggtcga gaccatcagc
     3181 cccggcgacg gccgcacctt tcccaagaga ggccagactt gcgtggtcca ctacaccggc
     3241 atgctggagg acggcaagaa gttcgacagc agccgcgacc gcaacaagcc cttcaagttc
     3301 atgctgggca aacaggaagt gatccgcggc tgggaggaag gcgtggctca gatgagcgtg
     3361 gggcagcggg ccaagctgac catcagcccc gactatgcct acggcgccac cggccacccc
     3421 ggcatcatcc ccccccacgc caccctggtg ttcgacgtgg agctgctgaa gctggagact
     3481 agtaccggtt ctagagtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     3541 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     3601 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     3661 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     3721 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     3781 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     3841 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3901 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3961 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     4021 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     4081 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     4141 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     4201 ctgtacaagt ccggactcag atctcgagct caagcttcga attctaccaa tgagatcttg
     4261 gactctgcct ctcacgactg ccccttagtc actcagacat acggcgcagc tgcaggaaaa
     4321 gcctacacag acgacctggg tgctgtgggt ggcgcctgcc tggaggacga ggccagcgcc
     4381 ctgcgcctgg atgaggacag cgagcacccg cccatgattc ttcggactga ctgaggatcc
     4441 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     4501 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     4561 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     4621 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     4681 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     4741 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     4801 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     4861 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     4921 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     4981 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgagatcct
     5041 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     5101 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     5161 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5221 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5281 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     5341 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     5401 agtgagaggc ccgggttaat taaggaaagg gctagatcat tcttgaagac gaaagggcct
     5461 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5521 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5581 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5641 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5701 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5761 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5821 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5881 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5941 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6001 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6061 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6121 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6181 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6241 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6301 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6361 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6421 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6481 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6541 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6601 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6661 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6721 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6781 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6841 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6901 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6961 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7021 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7081 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7141 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7201 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7261 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7321 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     7381 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     7441 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     7501 cagcaagctc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     7561 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     7621 ctccccgtgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     7681 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     7741 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac
     7801 gaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7861 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     7921 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     7981 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
     8041 aaacttagta gt
//
