LOCUS       sequence-447547-        8444 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 8444)
  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 8444)
  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 V5-NIX
FEATURES             Location/Qualifiers
     source          1..8444
                     /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             3288..3329
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4140..4163
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    4197..4770
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4220..4772
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4364..4381)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4591..4610
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4772..5371
                     /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(4772..4791)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5268..5288
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5427..6015
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5480..5500)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5898..5909)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6018..6034)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6019..6035)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6544..6724
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6786..7374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6866..6885)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7451..8242,8243..8311))
                     /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     8074..8093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8312..8416)
                     /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 attcatgggt aagcctatcc
     3301 ctaaccctct cctcggtctc gattctacgc gagttgcgcc cgggagatct gagctcccgc
     3361 ggtggcaggc accatatggt acccgtatgt cgtcccacct agtcgagccg ccgccgcccc
     3421 tgcacaacaa caacaacaac tgcgaggaaa atgagcagtc tctgcccccg ccggccggcc
     3481 tcaacagttc ctgggtggag ctacccatga acagcagcaa tggcaatgat aatggcaatg
     3541 ggaaaaatgg ggggctggaa cacgtaccat cctcatcctc catccacaat ggagacatgg
     3601 agaagattct tttggatgca caacatgaat caggacagag tagttccaga ggcagttctc
     3661 actgtgacag cccttcgcca caagaagatg ggcagatcat gtttgatgtg gaaatgcaca
     3721 ccagcaggga ccatagctct cagtcagaag aagaagttgt agaaggagag aaggaagtcg
     3781 aggctttgaa gaaaagtgcg gactgggtat cagactggtc cagtagaccc gaaaacattc
     3841 cacccaagga gttccacttc agacacccta aacgttctgt gtctttaagc atgaggaaaa
     3901 gtggagccat gaagaaaggg ggtattttct ccgcagaatt tctgaaggtg ttcattccat
     3961 ctctcttcct ttctcatgtt ttggctttgg ggctaggcat ctatattgga aagcgactga
     4021 gcacaccctc tgccagcacc tactgaggat ccggtaccga ggagatctgc cgccgcgatc
     4081 gccggcgcgc cagatctcaa gcttaactag ctagcggacc gacgcgtacg cggccgctcg
     4141 attacaagga tgacgacgat aaggtttaaa cctaggcgta gcggccgcaa attccgcccc
     4201 tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg
     4261 tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa
     4321 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg
     4381 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca
     4441 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc
     4501 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt
     4561 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg
     4621 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca
     4681 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac
     4741 gtggttttcc tttgaaaaac acgatgataa tatgaccgag tacaagccca cggtgcgcct
     4801 cgccacccgc gacgacgtcc cccgggcagt acgcaccctc gccgccgcgt tcgccgacta
     4861 ccccgccacg cgccacaccg tcgatccaga ccgccacatc gagcgggtca ccgagctgca
     4921 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     4981 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     5041 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     5101 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     5161 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     5221 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     5281 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     5341 ctggtgcatg acccgcaagc ccggtgcctg aacatgttta agggttccgg ttccactagg
     5401 tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     5461 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     5521 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     5581 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     5641 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     5701 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     5761 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     5821 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     5881 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     5941 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     6001 gggccgcctc cccgcatcga taccgtcgac ctcgatcgag acctagaaaa acatggagca
     6061 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag
     6121 gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag
     6181 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     6241 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     6301 gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc
     6361 tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc
     6421 cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag
     6481 tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt
     6541 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     6601 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     6661 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     6721 agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6781 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6841 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6901 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6961 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     7021 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     7081 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     7141 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     7201 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     7261 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     7321 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     7381 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     7441 ttggtcatga ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7501 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7561 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7621 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7681 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7741 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7801 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7861 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7921 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7981 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8041 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8101 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8161 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8221 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8281 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8341 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8401 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac
//
