LOCUS       sequence-448352-        7166 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 7166)
  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 7166)
  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_6xHis-TEV-mCherry-WIPI3
FEATURES             Location/Qualifiers
     source          1..7166
                     /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"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             134..838
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(276..294)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     544..563
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1982..2006
                     /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             2115..2159
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2197..2215)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2211..2258
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2295..2750
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2382..2401)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2592..2613
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2865..3656,3657..3725))
                     /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     3488..3507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3726..3818)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3899..4487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4388..4407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4641..4658
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4673..4812
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4738..4757)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4898..4920
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4914..5105)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5677..6759)
                     /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     6721..6740
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6760..6837)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7043..7062
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7150..7166,1..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 cggaaaacct
      121 gtattttcag ggagtgagca agggcgagga ggataacatg gccatcatca aggagttcat
      181 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
      241 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      301 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
      361 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
      421 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
      481 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
      541 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
      601 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
      661 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
      721 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
      781 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
      841 cggactcaga tctcgagctc aagcttcgat gaatctgctg ccttgtaatc cacacgggaa
      901 cgggctgctg tatgctgggt ttaatcagga ccacggctgc ttcgcttgcg gaatggagaa
      961 cgggttcaga gtgtacaata ccgacccact gaaagagaag gaaaaacagg agttcctgga
     1021 aggcggggtc ggacatgtgg aaatgctgtt taggtgcaac tacctggctc tggtgggagg
     1081 cgggaagaaa cccaagtatc cccctaacaa agtgatgatc tgggacgatc tgaagaaaaa
     1141 gactgtgatc gagattgagt tcagcaccga ggtcaaggcc gtgaaactgc ggagagaccg
     1201 catcgtggtc gtgctggatt ccatgatcaa ggtcttcacc tttacacaca accctcatca
     1261 gctgcacgtg ttcgaaacat gctataaccc caagggcctg tgcgtgctgt gccctaattc
     1321 aaacaatagc ctgctggctt ttcccggaac tcataccggc cacgtccagc tggtggacct
     1381 ggcatctact gagaagccac ccgtcgatat ccctgcacat gaaggggtgc tgtcctgtat
     1441 cgccctgaat ctgcagggaa cacgaattgc tactgcatct gagaagggca ccctgatccg
     1501 gattttcgac acaagctccg gccacctgat tcaggaactg aggcgcggga gtcaggccgc
     1561 taacatctac tgcattaact tcaatcagga cgccagcctg atctgcgtga gcagtgatca
     1621 tggcaccgtg cacatttttg cagccgagga tccaaagcga aacaagcagt caagcctggc
     1681 cagtgcttca ttcctgccca agtatttttc ctctaaatgg agcttctcca agtttcaggt
     1741 gccttctggg agtccatgca tctgtgcctt tggaacagag cctaacgcag tgatcgccat
     1801 ttgtgctgat ggcagctact ataaattcct gtttaatcca aagggggagt gtattaggga
     1861 tgtctatgct cagttcctgg agatgaccga cgataagctg taagcataat gcttaagtcg
     1921 aacagaaagt aatcgtattg tacacggccg cataatcgaa attaatacga ctcactatag
     1981 gggaattgtg agcggataac aattccccat cttagtatat tagttaagta taagaaggag
     2041 atatacatat ggcagatctc aattggatat cggccggcca cgcgatcgct gacgtcggta
     2101 ccctcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt
     2161 ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac
     2221 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2281 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2341 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2401 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2461 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2521 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2581 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2641 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2701 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt ctggcggcac
     2761 gatggcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2821 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2881 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2941 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3001 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3061 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3121 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3181 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3241 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3301 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3361 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3421 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3481 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3541 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3601 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3661 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3721 ctcatactct tcctttttca atcatgattg aagcatttat cagggttatt gtctcatgag
     3781 cggatacata tttgaatgta tttagaaaaa taaacaaata ggtcatgacc aaaatccctt
     3841 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3901 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3961 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4021 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4081 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4141 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4201 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4261 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4321 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4381 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4441 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4501 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4561 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4621 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4681 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4741 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4801 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4861 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4921 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4981 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5041 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5101 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5161 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5221 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5281 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5341 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5401 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5461 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5521 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
     5581 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5641 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5701 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5761 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5821 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5881 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5941 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6001 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6061 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6121 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6181 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6241 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6301 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6361 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6421 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6481 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6541 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6601 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6661 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6721 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6781 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6841 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6901 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6961 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     7021 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     7081 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
     7141 ccgcgaaatt aatacgactc actata
//
