LOCUS       sequence-447752-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant protein for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  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 6950)
  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: pETDuet1_FUNDC1(1-50aa)-EGFP-TEV-GST (Y18A/L21...
FEATURES             Location/Qualifiers
     source          1..6950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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)."
     CDS             join(456..458,459..1169)
                     /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(498..519)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(759..778)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1106..1127
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1182..1202
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1209..1226
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1233..1886
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1841..1863
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1899..1943
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1981..1999)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1995..2042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2079..2534
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2166..2185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2376..2397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2649..3440,3441..3509))
                     /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     3272..3291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3510..3602)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3683..4271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4172..4191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4425..4442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4457..4596
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4522..4541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4682..4704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4698..4889)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5461..6543)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6505..6524
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6544..6621)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6827..6846
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6934..6950,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tggcgacccg gaacccccct ccccaagact atgaaagtga tgacgactct gctgaagtgg
      361 cggatttaac tgagtatgca agaagacacc agtggtggaa tcgagtgttt ggccacagtt
      421 cgggacctat ggtagaaaaa tactcagtag gcggcgtgag caagggcgag gagctgttca
      481 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
      541 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
      601 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
      661 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
      721 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
      781 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
      841 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
      901 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
      961 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1021 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     1081 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1141 tcactctcgg catggacgag ctgtacaagg gcggcagtgg cgaaaacctg tattttcagg
     1201 gcggatcact ggttccgcgt ggatccggcg gatcccctat actaggttat tggaaaatta
     1261 agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc
     1321 atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg
     1381 agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca
     1441 tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag
     1501 agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat
     1561 atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga
     1621 aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc
     1681 ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg
     1741 atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata
     1801 agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg
     1861 gtggtggcga ccatcctcca aaatagctcg agtctggtaa agaaaccgct gctgcgaaat
     1921 ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca
     1981 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     2041 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     2101 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2161 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2221 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2281 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2341 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2401 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2461 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2521 aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat cttcacctag
     2581 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2641 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2701 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2761 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2821 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2881 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2941 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3001 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3061 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3121 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3181 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3241 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3301 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3361 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3421 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3481 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg attgaagcat
     3541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3601 aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3661 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3721 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3781 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3841 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3901 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3961 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4021 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4081 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4141 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4201 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4261 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4321 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4381 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4441 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4501 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4561 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4621 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4681 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4741 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4801 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4861 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4921 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4981 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5041 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5101 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5161 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5221 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5281 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5341 atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5401 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5461 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5521 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5581 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5641 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     5701 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     5761 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     5821 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5881 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5941 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6001 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6061 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6121 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6181 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6241 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6301 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6361 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6421 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6481 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6541 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6601 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6661 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     6721 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6781 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6841 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6901 agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata
//
