LOCUS       sequence-447441-        6125 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 6125)
  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 6125)
  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_His-TEV-EGFP
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        148..166
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    167..191
                     /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             206..228
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             247..264
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             283..303
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(304..306,307..1020)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(304..306,307..1017)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(346..367)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(607..626)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     954..975
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1086..1104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1105..1129
                     /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             1238..1282
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1320..1338)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1334..1381
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1418..1873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1505..1524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1715..1736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1988..2779,2780..2848))
                     /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     2611..2630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2849..2941)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3022..3610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3511..3530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3764..3781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3796..3935
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3861..3880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4021..4043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4037..4228)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4800..5882)
                     /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     5844..5863
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5883..5960)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
       61 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      121 tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
      181 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
      241 agcagccatc accatcatca ccacagccag gatccgaatt cggaaaacct gtattttcag
      301 ggagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      361 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      421 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      481 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      541 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      601 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      661 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      721 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      781 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      841 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      901 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      961 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga
     1021 gcggccgcat aatgcttaag tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc
     1081 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc catcttagta
     1141 tattagttaa gtataagaag gagatataca tatggcagat ctcaattgga tatcggccgg
     1201 ccacgcgatc gctgacgtcg gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt
     1261 tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac
     1321 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1381 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1441 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1501 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1561 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1621 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1681 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1741 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1801 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1861 acgtttacaa tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga
     1921 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     1981 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2041 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2101 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2161 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2221 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2281 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     2521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     2581 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     2641 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     2701 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     2761 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     2821 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt
     2881 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2941 ataggtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3001 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3061 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3121 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3181 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3241 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3301 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3361 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3421 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3481 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3541 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3601 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3661 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3721 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3781 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3841 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3901 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3961 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4021 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4081 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4141 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4201 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4261 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4321 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4381 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4441 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4501 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4561 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4621 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4681 tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4741 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4801 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4861 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4921 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4981 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5041 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
     5101 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5161 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5221 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5281 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5341 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5401 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5461 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5521 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5581 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5641 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5701 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5761 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5821 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5881 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5941 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6001 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6061 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6121 acaag
//
