LOCUS       sequence-447442-        6635 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 6635)
  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 6635)
  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-EGFP-FIP200-CTR(1429-1591aa)
FEATURES             Location/Qualifiers
     source          1..6635
                     /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             119..139
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(140..142,143..853)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(182..203)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(443..462)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     790..811
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1432..1450
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1451..1475
                     /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             1584..1628
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1666..1684)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1680..1727
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1764..2219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1851..1870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2061..2082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2334..3125,3126..3194))
                     /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     2957..2976
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3195..3287)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3368..3956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3857..3876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4110..4127
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4142..4281
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4207..4226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4367..4389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4383..4574)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5146..6228)
                     /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     6190..6209
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6229..6306)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6512..6531
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6619..6635,1..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 cgaattcgga
      121 aaacctgtat tttcagggag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      181 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      241 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      301 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      361 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      421 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
      481 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
      541 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
      601 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
      661 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
      721 gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa
      781 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
      841 cgagctgtac aagtccggac tcagatctcg aggcgcagat gaaggaagag tggattcagc
      901 aatggagaca agcatgatgt ctgtacaaga aaatattcat atgttgtctg aagaaaaaca
      961 gcggataatg ctgttagaac gaacattgca attgaaagaa gaagaaaata aacggttaaa
     1021 tcaaagactg atgtctcaga gcatgtcttc agtatcttca aggcattctg aaaagatagc
     1081 tattagagat tttcaggtgg gagatttggt actcatcatc ctagacgaac gccatgacaa
     1141 ttatgtgtta tttactgtta gtcctacttt atattttcta cattcagagt ctctacctgc
     1201 cctggatctc aaaccagctt caggtgcatc tagaagaccc tgggtacttg gaaaagtaat
     1261 ggaaaaagaa tactgtcaag ccaaaaaggc acaaaacaga tttaaagttc ctttggggac
     1321 aaagttttac agagtgaaag ccgtatcatg gaataagaaa gtatgagcgg ccgcataatg
     1381 cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac
     1441 tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat
     1501 aagaaggaga tatacatatg gcagatctca attggatatc ggccggccac gcgatcgctg
     1561 acgtcggtac cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     1621 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     1681 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1741 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1801 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1861 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1921 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1981 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2041 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2101 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2161 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2221 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2281 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2341 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2401 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2461 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2521 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2581 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2641 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2701 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2761 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2821 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2881 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2941 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3001 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3061 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3121 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3181 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     3241 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     3301 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3361 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3421 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3481 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3541 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3601 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3661 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3721 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3781 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3841 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3901 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3961 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4021 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4081 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4141 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     4201 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4261 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4321 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4381 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4441 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4501 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4561 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4621 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4681 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4741 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4801 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4861 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4921 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4981 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
     5041 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5101 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5161 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5221 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5281 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5341 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5401 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     5461 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5521 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5581 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5641 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5701 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5761 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5821 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5881 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5941 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6001 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6061 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6121 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6181 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6241 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6301 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6361 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6421 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6481 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6541 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     6601 atctcgatcc cgcgaaatta atacgactca ctata
//
