LOCUS       sequence-462157-        9186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of protein in mammalian cell culture, rescue 
            experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9186)
  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 9186)
  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: pGenlenti_ATG13-WT
FEATURES             Location/Qualifiers
     source          1..9186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /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    2443..2560
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2643..3022
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3023..3226
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3252..3271
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3252..3270
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4882..4905
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    4939..5512
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4962..5514
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5106..5123)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5333..5352
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5514..6113
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5514..5533)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6010..6030
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    6169..6757
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6222..6242)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6640..6651)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6760..6776)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6761..6777)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7286..7466
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7528..8116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7608..7627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8193..8984,8985..9053))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8816..8835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9054..9158)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2581 cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg
     2641 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2701 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2761 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2821 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2881 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
     2941 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
     3001 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata
     3061 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3121 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca
     3181 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     3241 tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccatg
     3301 gaaactgatc tcaattccca ggacagaaag gacctggaca agtttattaa attttttgcc
     3361 ctcaagactg tccaagtgat tgtccaggct cggcttggtg aaaagatttg cactcgttca
     3421 tcatcttctc caacgggttc agattggttc aacttagcaa tcaaagacat cccagaggtt
     3481 acacatgaag caaagaaggc actggcagga cagctgcctg cagtcgggag gtccatgtgt
     3541 gtggagattt cacttaagac ttctgaggga gattccatgg agctggaaat atggtgtctt
     3601 gaaatgaatg aaaagtgtga taaagaaatc aaagtttcct acacggtgta caacagactg
     3661 tcattgctgc tgaagtccct tcttgctata actagggtga caccagccta taggctctcc
     3721 aggaaacaag ggcatgaata tgtcatatta tacaggatat attttggaga agttcagctg
     3781 agtggcttag gagaaggctt ccagacagtt cgtgttggga cagtgggcac ccctgtgggc
     3841 accatcactc tttcttgtgc ttacagaatt aacttggcat tcatgtctac caggcaattt
     3901 gagaggaccc cacctatcat ggggattatt attgatcact ttgtggaccg tccctatccc
     3961 agctcctctc ccatgcaccc ctgcaattac agaactgctg gtgaggacac tggagtaata
     4021 tacccgtctg tagaagactc tcaagaagtg tgtaccacct ctttttccac ctccccacca
     4081 tcccagctct caagctctcg cctttcctat cagcctgctg ccctgggcgt tggatcagct
     4141 gacctggctt atccagtagt gtttgctgct ggcttaaatg ctacacaccc tcaccagctg
     4201 atggttcctg ggaaggaagg tggggtaccc cttgctccca accagcctgt ccatggtacc
     4261 caggctgacc aggagagact ggcaacctgc accccttctg acagaaccca ctgtgctgcc
     4321 acaccctcca gtagtgagga tactgaaacc gtatcaaaca gcagtgaggg acgggcctcc
     4381 cctcacgatg tcttggagac catctttgtc cgaaaagtgg gggcttttgt caacaaaccc
     4441 attaaccagg tgaccctgac gagtttggat ataccctttg ccatgtttgc tcccaagaat
     4501 ttggagctgg aggataccga tccaatggtg aatcctccag attccccaga gactgaatct
     4561 cctctccagg gcagcctgca ctcagatggc tccagcgggg gcagcagtgg caatacccat
     4621 gatgactttg ttatgataga ctttaaacca gctttttcta aagatgacat tcttccgatg
     4681 gacctgggga ccttctatcg ggagtttcag aacccacctc agctgagcag cctctccata
     4741 gatattggag cacagtccat ggctgaagac ttggactcat taccagagaa gctggctgtg
     4801 catgagaaga atgtccgcga gtttgatgcc tttgtggaaa ccctgcagta agctagcgga
     4861 ccgacgcgta cgcggccgct cgattacaag gatgacgacg ataaggttta aacctaggcg
     4921 tagcggccgc aaattccgcc cctctccctc ccccccccct aacgttactg gccgaagccg
     4981 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     5041 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     5101 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     5161 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     5221 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     5281 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     5341 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     5401 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     5461 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatgaccg
     5521 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggca gtacgcaccc
     5581 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatcca gaccgccaca
     5641 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     5701 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     5761 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     5821 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     5881 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     5941 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     6001 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     6061 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacatgtt
     6121 taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg
     6181 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     6241 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     6301 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     6361 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     6421 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     6481 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     6541 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     6601 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     6661 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     6721 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg
     6781 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg
     6841 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc
     6901 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     6961 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta
     7021 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata
     7081 tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga
     7141 agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga
     7201 cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc
     7261 ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct gagcctggga
     7321 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     7381 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     7441 ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa
     7501 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     7561 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     7621 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     7681 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     7741 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     7801 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     7861 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     7921 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     7981 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     8041 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     8101 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     8161 cgaaaactca cgttaaggga ttttggtcat gattaccaat gcttaatcag tgaggcacct
     8221 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     8281 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     8341 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     8401 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     8461 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     8521 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     8581 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     8641 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     8701 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     8761 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     8821 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     8881 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8941 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     9001 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     9061 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     9121 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     9181 cctgac
//
