LOCUS       sequence-430432-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression in E. coli of OPTN (Internal ref. SMC1789).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  AUTHORS   Adriaenssens E, Nguyen TN, Sawa-Makarska J, Khuu G, Schuschnig M, 
            Shoebridge S, Watts EM, Csalyi KD, Padman BS, Lazarou M, Martens S
  TITLE     Control of mitophagy initiation and progression by the TBK1 adaptors
            NAP1 and SINTBAD
  JOURNAL   bioRxiv September 25, 202
REFERENCE   2  (bases 1 to 7778)
  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: "bioRxiv 
            September 25, 202"
COMMENT     Sequence Label: pET-Duet_OPTN-thrombin-GST
FEATURES             Location/Qualifiers
     source          1..7778
                     /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"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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             617..628
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2037..2054
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2061..2714
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2669..2691
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2727..2771
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2809..2827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2823..2870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2907..3362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2994..3013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3204..3225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3477..4268,4269..4337))
                     /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     4100..4119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4338..4430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4511..5099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5000..5019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5253..5270
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5285..5424
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5350..5369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5510..5532
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5526..5717)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6289..7371)
                     /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     7333..7352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7372..7449)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7655..7674
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7762..7778,1..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 cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgtcccatca acctctcagc tgcctcactg aaaaggagga cagccccagt gaaagcacag
      361 gaaatggacc cccccacctg gcccacccaa acctggacac gtttaccccg gaggagctgc
      421 tgcagcagat gaaagagctc ctgaccgaga accaccagct gaaagaagcc atgaagctaa
      481 ataatcaagc catgaaaggg agatttgagg agctttcggc ctggacagag aaacagaagg
      541 aagaacgcca gttttttgag atacagagca aagaagcaaa agagcgtcta atggccttga
      601 gtcatgagaa tgagaaattg aaggaagagc ttggaaaact aaaagggaaa tcagaaaggt
      661 catctgagga ccccactgat gactccaggc ttcccagggc cgaagcggag caggaaaagg
      721 accagctcag gacccaggtg gtgaggctac aagcagagaa ggcagacctg ttgggcatcg
      781 tgtctgaact gcagctcaag ctgaactcca gcggctcctc agaagattcc tttgttgaaa
      841 ttaggatggc tgaaggagaa gcagaagggt cagtaaaaga aatcaagcat agtcctgggc
      901 ccacgagaac agtctccact ggcacggcat tgtctaaata taggagcaga tctgcagatg
      961 gggccaagaa ttacttcgaa catgaggagt taactgtgag ccagctcctg ctgtgcctaa
     1021 gggaagggaa tcagaaggtg gagagacttg aagttgcact caaggaggcc aaagaaagag
     1081 tttcagattt tgaaaagaaa acaagtaatc gttctgagat tgaaacccag acagagggga
     1141 gcacagagaa agagaatgat gaagagaaag gcccggagac tgttggaagc gaagtggaag
     1201 cactgaacct ccaggtgaca tctctgttta aggagcttca agaggctcat acaaaactca
     1261 gcgaagctga gctaatgaag aagagacttc aagaaaagtg tcaggccctt gaaaggaaaa
     1321 attctgcaat tccatcagag ttgaatgaaa agcaagagct tgtttatact aacaaaaagt
     1381 tagagctaca agtggaaagc atgctatcag aaatcaaaat ggaacaggct aaaacagagg
     1441 atgaaaagtc caaattaact gtgctacaga tgacacacaa caagcttctt caagaacata
     1501 ataatgcatt gaaaacaatt gaggaactaa caagaaaaga gtcagaaaaa gtggacaggg
     1561 cagtgctgaa ggaactgagt gaaaaactgg aactggcaga gaaggctctg gcttccaaac
     1621 agctgcaaat ggatgaaatg aagcaaacca ttgccaagca ggaagaggac ctggaaacca
     1681 tgaccatcct cagggctcag atggaagttt actgttctga ttttcatgct gaaagagcag
     1741 cgagagagaa aattcatgag gaaaaggagc aactggcatt gcagctggca gttctgctga
     1801 aagagaatga tgctttcgaa gacggaggca ggcagtcctt gatggagatg cagagtcgtc
     1861 atggggcgag aacaagtgac tctgaccagc aggcttacct tgttcaaaga ggagctgagg
     1921 acagggactg gcggcaacag cggaatattc cgattcattc ctgccccaag tgtggagagg
     1981 ttctgcctga catagacacg ttacagattc acgtgatgga ttgcatcatt ggatcactgg
     2041 ttccgcgtgg atccggcgga tcccctatac taggttattg gaaaattaag ggccttgtgc
     2101 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
     2161 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
     2221 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
     2281 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
     2341 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
     2401 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
     2461 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
     2521 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
     2581 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
     2641 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
     2701 atcctccaaa atagctcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc
     2761 acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat
     2821 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2881 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2941 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3001 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3061 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3121 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3181 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3241 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3301 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3361 ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3421 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3481 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3541 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3601 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3661 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3721 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3781 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3841 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3901 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3961 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4021 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4081 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4141 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4201 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4261 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4321 aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt atcagggtta
     4381 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggtcatga
     4441 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4501 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4561 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4621 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4681 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4741 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4801 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4861 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4921 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4981 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5041 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5101 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5161 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5221 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5281 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     5341 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     5401 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     5461 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5521 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     5581 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     5641 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     5701 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     5761 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     5821 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     5881 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5941 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     6001 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     6061 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     6121 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc
     6181 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     6241 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     6301 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6361 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     6421 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6481 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6541 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     6601 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6661 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6721 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6781 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6841 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6901 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6961 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     7021 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     7081 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     7141 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     7201 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     7261 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     7321 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     7381 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     7441 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     7501 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     7561 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     7621 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     7681 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     7741 tcgatctcga tcccgcgaaa ttaatacgac tcactata
//
