LOCUS       sequence-477070-        4370 bp    DNA     circular SYN 25-JUN-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 4370)
  AUTHORS   Adriaenssens E, Schaar S, Cook ASI, Stuke JFM, Sawa-Makarska J, 
            Nguyen TN, Ren X, Schuschnig M, Romanov J, Khuu G, Lazarou M, Hummer
            G, Hurley JH, Martens S
  TITLE     Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways 
            and hierarchical flexibility of the initiation machinery.
  JOURNAL   bioRxiv [Preprint]. 2024 Aug 28:2024.08.28.609967. doi: 
            10.1101/2024.08.28.609967.
  PUBMED    39253418
REFERENCE   2  (bases 1 to 4370)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Aug 28:2024.08.28.609967. doi: 
            10.1101/2024.08.28.609967."
COMMENT     Sequence Label: pGenLenti V5-BNIP3
FEATURES             Location/Qualifiers
     source          1..4370
                     /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"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     terminator      complement(1070..1156)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1799..1818
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(1928..2022)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2056..2763)
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(2092..2113)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2700..2721
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     RBS             3031..3042
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    3061..3077
                     /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)."
     promoter        complement(join(3085..3091,3092..3108,3109..3114,
                     3115..3129))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3093..3109
                     /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     complement(3153..3171)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3235..3339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3340..3408,3409..4200)
                     /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     complement(3558..3577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct 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 tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctct agagggcgga tttgtcctac
     1081 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag
     1141 cctttcgttt tatttgatgc ctcaagctag agagtcatta ccccaggcgt ttaagggcac
     1201 caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
     1261 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     1321 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatgcatgg tgaaaacggg
     1381 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     1441 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     1501 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     1561 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     1621 aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt ccggatgagc
     1681 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     1741 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     1801 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     1861 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgccaagct
     1921 agcttggatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca
     1981 gatggagttc tgaggtcatt actggatcta tcaacaggag tccaagctca gctaattaag
     2041 cttgcggccg ctttaagaac cgccactgcc tccagtgatc ccggcggcgg tcacgaactc
     2101 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagcttgg actgggtgct
     2161 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
     2221 gtggtcggcg agctgcacgc tgccgtcctc cacgttgtgg cggatcttga agttggcctt
     2281 gatgccgttc ttctgcttgt cggcggtgat atagacgttg tggctgttgt agttgtactc
     2341 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
     2401 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagg tgccgtcgtc
     2461 cttgaaggtg atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
     2521 gtgctgcttc atgtggtcgg ggtagcggct gaagctctgc acgccgtagg tcagggtggt
     2581 cacgagggtg ggccagggca cgggcagctt gccggtggtg ctgatgaact tcagggtcag
     2641 cttgccgttg gtggcatcgc cctcgccctc gccgcgcacg ctgaacttgt ggccgtttac
     2701 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacaggctct cagtgctatc
     2761 catggtgcag tagttctcca gctggtagag ggagcagatg ctggtacagc attgttccac
     2821 aatgccacgc ttctgcaggg acccctccag ggccaagggc tgcaggctgc ctgcaccagg
     2881 gcccccgccc agctccacct gccccacctg caggtcctct gcctcccggc gggtcttggg
     2941 tgtgtagaag aagcctcgtt ccccgcacac taggtagaga gcttccacca ggtgtgagcc
     3001 gcacaggtgt tggttcacaa acatggttaa tttctcctct ttaatgaatt ctgtgtgaaa
     3061 ttgttatccg ctcacaattg aatctattat aattgttatc cgctcacaaa gcaaataaat
     3121 tttttatgat ttctcgaggt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     3181 taatgtcatg ataataatgg tttcttagca ggtggcactt ttcggggaaa tgtgcgcgga
     3241 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3301 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3361 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3421 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3481 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3541 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3601 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3661 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3721 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3781 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3841 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3901 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3961 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4021 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4081 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4141 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4201 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
