LOCUS       sequence-462273-        6332 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 6332)
  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 6332)
  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-WIPI2d IDR (364-425aa)
FEATURES             Location/Qualifiers
     source          1..6332
                     /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        1129..1147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1148..1172
                     /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             1281..1325
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1363..1381)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1377..1424
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1461..1916
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1548..1567)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1758..1779
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2031..2822,2823..2891))
                     /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     2654..2673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2892..2984)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3065..3653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3554..3573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3807..3824
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3839..3978
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3904..3923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4064..4086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4080..4271)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4843..5925)
                     /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     5887..5906
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5926..6003)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6209..6228
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6316..6332,1..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 aagcttcgaa ttcgaccaat gagatcttgg actctgcctc
      901 tcacgactgc cccttagtca ctcagacata cggcgcagct gcaggaaaag cctacacaga
      961 cgacctgggt gctgtgggtg gcgcctgcct ggaggacgag gccagcgccc tgcgcctgga
     1021 tgaggacagc gagcacccgc ccatgattct tcggactgac tgagcggccg cataatgctt
     1081 aagtcgaaca gaaagtaatc gtattgtaca cggccgcata atcgaaatta atacgactca
     1141 ctatagggga attgtgagcg gataacaatt ccccatctta gtatattagt taagtataag
     1201 aaggagatat acatatggca gatctcaatt ggatatcggc cggccacgcg atcgctgacg
     1261 tcggtaccct cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
     1321 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
     1381 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1441 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1501 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1561 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1621 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1681 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1741 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1801 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1861 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttctgg
     1921 cggcacgatg gcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1981 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2041 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2101 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2161 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2221 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2281 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2341 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2401 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2461 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2521 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2581 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2641 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2701 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2761 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2821 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2881 tgaatactca tactcttcct ttttcaatca tgattgaagc atttatcagg gttattgtct
     2941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggtc atgaccaaaa
     3001 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3061 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3121 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3181 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3241 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3301 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3361 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3421 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3481 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3541 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3601 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3661 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3721 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3781 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3841 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3901 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3961 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4021 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4081 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4141 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4201 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4261 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4321 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4381 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4441 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4501 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4561 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4621 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4681 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct agtcatgccc
     4741 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4801 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4861 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4921 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4981 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     5041 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     5101 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     5161 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5221 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5281 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5341 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5401 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5461 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5521 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5581 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5641 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5701 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5761 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5821 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5881 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5941 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     6001 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     6061 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     6121 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     6181 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     6241 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc
     6301 tcgatcccgc gaaattaata cgactcacta ta
//
