LOCUS       sequence-448392-        7101 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 7101)
  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 7101)
  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: pCAG_mCherry-ATG13 (WT)
FEATURES             Location/Qualifiers
     source          1..7101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          277..1293
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1217..1239
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1301..1320
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1361..2068
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1506..1524)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1774..1793
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2598..2609
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(3736..3755)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3801..3856
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3855..3874)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4217..4233)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4217..4233)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4230..4252)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4241..4257
                     /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)."
     promoter        complement(join(4265..4271,4272..4289,4290..4295))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4310..4331
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4390..4585
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4436..4571
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4591..4725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4628..4647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4682..4701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4793..4810)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4964..5552)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5044..5063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5723..6514,6515..6583))
                     /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     6346..6365
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6584..6688)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6719..7098
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7100..7101,1..276)
                     /label=chicken beta-actin promoter
ORIGIN
        1 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
       61 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      121 sgcgcgccgg gggggggggg ggggggsggg gggsggggsg gggsgrggcg gagaggtgcg
      181 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      241 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      301 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      361 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      421 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      481 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      541 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
      601 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
      661 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
      721 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
      781 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
      841 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggrgggc
      901 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
      961 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1021 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1081 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1141 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1201 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1261 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
     1321 ctgtctcatc attttggcaa agaattcctc gagccaccat atggtgagca agggcgagga
     1381 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     1441 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     1501 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     1561 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     1621 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     1681 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     1741 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     1801 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     1861 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     1921 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     1981 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     2041 caccggcggc atggacgagc tgtacaaggg taccgagacc gacctcaaca gccaggaccg
     2101 taaagacctc gacaagttca ttaagttttt cgccctcaag accgtgcagg tgatcgtgca
     2161 ggcccggctg ggagagaaaa tctgcacacg cagctcctct tcacccaccg gttctgactg
     2221 gttcaacctg gctattaagg atatccctga agtgactcac gaagcaaaga aagcactcgc
     2281 tggacagctg ccagcagtgg gtcgttcaat gtgtgtcgag atctccctga agacatctga
     2341 aggagactca atggagctcg aaatttggtg cctggagatg aacgaaaagt gtgataaaga
     2401 gatcaaagtg agctacaccg tctataatcg tctgtccctg ctcctgaaat ctctcctggc
     2461 catcactcgg gtgacacctg cttaccgcct cagtaggaag cagggtcatg aatatgtgat
     2521 tctgtacagg atctatttcg gcgaggtcca gctctccgga ctgggtgaag gctttcagac
     2581 tgtgcgtgtc ggaaccgtgg gaactccagt cggcacaatt accctctctt gcgcataccg
     2641 tatcaatctg gccttcatgt caaccagaca gtttgagcgt actcccccta ttatgggaat
     2701 cattatcgac cacttcgtgg atagaccata ccccagtagc tcccctatgc atccatgcaa
     2761 ctatcgtaca gccggtgaag acaccggcgt gatctacccc agtgtcgagg atagccagga
     2821 agtgtgtacc acttccttct ctacctcacc accctcacag ctctcttcaa gtcgcctgag
     2881 ttatcagcct gccgctctcg gagtcggtag cgcagacctg gcctacccag tggtcttcgc
     2941 agcaggcctc aacgcaaccc acccacatca gctgatggtg ccaggaaagg agggtggagt
     3001 cccactggct cctaatcagc cagtgcacgg tactcaggct gaccaggaaa ggctggcaac
     3061 ttgcacacca tccgatagaa ctcattgtgc tgcaacaccc agctcctctg aggacaccga
     3121 aactgtgagc aactcaagtg agggtcgcgc ctccccacac gatgtcctgg aaaccatctt
     3181 cgtgagaaaa gtcggcgctt ttgtgaacaa gcccattaat caggtcacac tcacctctct
     3241 ggacatccct ttcgctatgt ttgcaccaaa gaacctcgag ctggaagaca ctgatcccat
     3301 ggtgaatcct ccagactcac ccgagacaga aagtcctctc cagggcagtc tgcacagcga
     3361 tggaagctcc ggtggctctt caggcaacac acatgacgat ttcgtcatga ttgacttcaa
     3421 acccgctttt tccaaggacg atatcctccc tatggatctg ggaaccttct acagggagtt
     3481 tcagaatccc cctcagctca gtagcctgag cattgacatc ggtgcccagt ccatggctga
     3541 ggacctcgat tctctgcctg aaaaactggc tgtgcacgag aagaacgtca gagaattcga
     3601 cgcatttgtg gagaccctgc agtgacgcca ttaacctgat gttctggggc aagcttacta
     3661 gtcctcagtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     3721 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     3781 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     3841 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     3901 tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg
     3961 ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca
     4021 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc
     4081 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg
     4141 actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa
     4201 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4261 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4321 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4381 agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4441 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4501 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4561 ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac
     4621 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4681 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa
     4741 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4801 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4861 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4921 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4981 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5041 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5101 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5161 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5221 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5281 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5341 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5401 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5461 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5521 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5581 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5641 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5701 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5761 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5821 gggagggctt accatctggc cccagtgctg caatgatacc tcgagagcca cgctcaccgg
     5881 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5941 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6001 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6061 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6121 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6181 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6241 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6301 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6361 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6421 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6481 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6541 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6601 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6661 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtcga
     6721 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     6781 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     6841 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     6901 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     6961 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     7021 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     7081 gtcatcgcta ttaccatggt c
//
