LOCUS       sequence-448195-        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of protein in mammalian cell culture.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6918)
  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 6918)
  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-FRB-Fis1 (no mt-mKeima)
FEATURES             Location/Qualifiers
     source          1..6918
                     /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"
     CDS             2827..3105
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     misc_feature    3308..3896
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3361..3381)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3779..3790)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3971..4204
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4323..4341)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4409..4513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4514..4582,4583..5374)
                     /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(4732..4751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5545..6133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6034..6053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6287..6304
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6544..6565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6580..6585,6586..6603,6604..6610)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6618..6634
                     /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     6623..6645
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6642..6658
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6642..6658
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6708..6727
                     /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 ccgcatggct
     2821 tctagaatcc tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
     2881 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
     2941 gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
     3001 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc
     3061 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagactag ttacgtccgc
     3121 gggttgctgc agacagagcc ccagaacaac caggccaagg aactggagcg gctcattgac
     3181 aaggccatga agaaagatgg actcgtgggc atggccatcg tgggaggcat ggccctgggt
     3241 gtggcgggac tggccggact catcggactt gctgtgtcca agtccaaatc ctgaacgcgt
     3301 ggatcctaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3361 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3421 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3481 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3541 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3601 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3661 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3721 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3781 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3841 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctga
     3901 gatcctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     3961 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     4021 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4081 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     4141 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     4201 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4261 tcagagagtg agaggcccgg gttaattaag gaaagggcta gatcattctt gaagacgaaa
     4321 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4381 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4441 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4501 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4561 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4621 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4681 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4741 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     4801 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4861 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4921 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4981 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5041 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5101 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5161 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5221 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5281 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5341 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5401 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5461 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5521 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5581 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5641 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5701 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5761 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5821 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5881 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5941 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6001 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6061 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6121 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6181 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6241 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6301 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     6361 ttaatgcagc aagctcatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     6421 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     6481 aaaaagctcc ccgtggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     6541 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     6601 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat
     6661 gattacgaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6721 actcatcaat gtatcttatc atgtctggat caactggata actcaagcta accaaaatca
     6781 tcccaaactt cccaccccat accctattac cactgccaat tacctgtggt ttcatttact
     6841 ctaaacctgt gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg
     6901 tactacaaac ttagtagt
//
