LOCUS       sequence-448351-        7115 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 7115)
  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 7115)
  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-ATG13 IDR (191-517aa)
FEATURES             Location/Qualifiers
     source          1..7115
                     /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        1912..1930
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1931..1955
                     /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             2064..2108
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2146..2164)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2160..2207
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2244..2699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2331..2350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2541..2562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2814..3605,3606..3674))
                     /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     3437..3456
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3675..3767)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3848..4436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4337..4356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4590..4607
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4622..4761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4687..4706)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4847..4869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4863..5054)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5626..6708)
                     /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     6670..6689
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6709..6786)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6992..7011
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7099..7115,1..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 caatctggcc ttcatgtcaa ccagacagtt
      901 tgagcgtact ccccctatta tgggaatcat tatcgaccac ttcgtggata gaccataccc
      961 cagtagctcc cctatgcatc catgcaacta tcgtacagcc ggtgaagaca ccggcgtgat
     1021 ctaccccagt gtcgaggata gccaggaagt gtgtaccact tccttctcta cctcaccacc
     1081 ctcacagctc tcttcaagtc gcctgagtta tcagcctgcc gctctcggag tcggtagcgc
     1141 agacctggcc tacccagtgg tcttcgcagc aggcctcaac gcaacccacc cacatcagct
     1201 gatggtgcca ggaaaggagg gtggagtccc actggctcct aatcagccag tgcacggtac
     1261 tcaggctgac caggaaaggc tggcaacttg cacaccatcc gatagaactc attgtgctgc
     1321 aacacccagc tcctctgagg acaccgaaac tgtgagcaac tcaagtgagg gtcgcgcctc
     1381 cccacacgat gtcctggaaa ccatcttcgt gagaaaagtc ggcgcttttg tgaacaagcc
     1441 cattaatcag gtcacactca cctctctgga catccctttc gctatgtttg caccaaagaa
     1501 cctcgagctg gaagacactg atcccatggt gaatcctcca gactcacccg agacagaaag
     1561 tcctctccag ggcagtctgc acagcgatgg aagctccggt ggctcttcag gcaacacaca
     1621 tgacgatttc gtcatgattg acttcaaacc cgctttttcc aaggacgata tcctccctat
     1681 ggatctggga accttctaca gggagtttca gaatccccct cagctcagta gcctgagcat
     1741 tgacatcggt gcccagtcca tggctgagga cctcgattct ctgcctgaaa aactggctgt
     1801 gcacgagaag aacgtcagag aattcgacgc atttgtggag accctgcagt gagcataatg
     1861 cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac
     1921 tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat
     1981 aagaaggaga tatacatatg gcagatctca attggatatc ggccggccac gcgatcgctg
     2041 acgtcggtac cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     2101 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     2161 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2221 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2281 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2341 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2401 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2461 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2521 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2581 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2641 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2701 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2761 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2821 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2881 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2941 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3001 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3061 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3121 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3181 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3241 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3301 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3361 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3421 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3481 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3541 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3601 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3661 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     3721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     3781 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3841 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3901 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3961 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4021 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4081 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4141 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4201 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4261 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4321 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4381 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4441 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4501 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4561 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4621 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     4681 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4741 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4801 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4861 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4921 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4981 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     5041 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     5101 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     5161 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     5221 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     5281 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     5341 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     5401 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     5461 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
     5521 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5581 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5641 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5701 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5761 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5821 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5881 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     5941 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     6001 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     6061 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     6121 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     6181 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     6241 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     6301 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6361 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6421 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6481 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6541 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6601 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6661 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6721 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6781 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6841 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6901 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6961 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     7021 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     7081 atctcgatcc cgcgaaatta atacgactca ctata
//
