LOCUS       sequence-391289-        8597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for the expression and purification of EGFP labelled TBK1 
            carrying a mutation for the removal of the CTD (SMC Internal No. 
            1874).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8597)
  AUTHORS   Nguyen TN, Sawa-Makarska J, Khuu G, Lam WK, Adriaenssens E, 
            Fracchiolla D, Shoebridge S, Bernklau D, Padman BS, Skulsuppaisarn 
            M, Lindblom RSJ, Martens S, Lazarou M
  TITLE     Unconventional initiation of PINK1/Parkin mitophagy by Optineurin.
  JOURNAL   Mol Cell. 2023 May 18;83(10):1693-1709.e9. doi: 
            10.1016/j.molcel.2023.04.021.
  PUBMED    37207627
REFERENCE   2  (bases 1 to 8597)
  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"; doi: 
            "10.1016/j.molcel.2023.04"; journalName: "Mol Cell"; date: 
            "2023-05-18- 18"; volume: "83"; issue: "10"; pages: "1693-1709.e9"
COMMENT     Sequence Label: pFastBac_Dual_GST-TEV-EGFP-TBK1-Delta-CTD (res 
            1-666)
FEATURES             Location/Qualifiers
     source          1..8597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     265..286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        449..553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(554..622,623..1414)
                     /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     complement(772..791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1585..2173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2074..2093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2327..2344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2478..2702
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2769..3302)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3491..3496,3497..3513,3514..3519))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4027..4075
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4100..4119
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4211..4320)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4344..4435
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4360..4377
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             4478..5134
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     5092..5114
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             5141..5158
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5168..5188
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(5195..5197,5198..5200,5201..5911)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5240..5261)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5501..5520)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5848..5869
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    8054..8188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8091..8110)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8145..8164
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  8217..8382
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      join(8566..8597,1..423)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
       61 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      121 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      181 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      241 acgttggagt ccacgttctt aatagtggac tcttgttcca aactggaaca acactcaacc
      301 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
      361 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
      421 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      481 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
      541 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
      601 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
      661 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
      721 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
      781 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
      841 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
      901 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
      961 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1021 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1081 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1141 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1201 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1261 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1321 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     1381 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     1441 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1501 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1561 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1621 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1681 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1741 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1801 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1861 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1921 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1981 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2041 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2101 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2161 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2221 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2281 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2341 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2401 acaccgcata gaccagccgc gtaacctggc aaaatcggtt acggttgagt aataaatgga
     2461 tgccctgcgt aagcgggtgt gggcggacaa taaagtctta aactgaacaa aatagatcta
     2521 aactatgaca ataaagtctt aaactagaca gaatagttgt aaactgaaat cagtccagtt
     2581 atgctgtgaa aaagcatact ggacttttgt tatggctaaa gcaaactctt cattttctga
     2641 agtgcaaatt gcccgtcgta ttaaagaggg gcgtggccaa gggcatggta aagactatat
     2701 tcgcggcgtt gtgacaattt accgaacaac tccgcggccg ggaagccgat ctcggcttga
     2761 acgaattgtt aggtggcggt acttgggtcg atatcaaagt gcatcacttc ttcccgtatg
     2821 cccaactttg tatagagagc cactgcggga tcgtcaccgt aatctgcttg cacgtagatc
     2881 acataagcac caagcgcgtt ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg
     2941 ccctgcctcc ggtgctcgcc ggagactgcg agatcataga tatagatctc actacgcggc
     3001 tgctcaaact tgggcagaac gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag
     3061 gcagcaagcg cgatgaatgt cttactacgg agcaagttcc cgaggtaatc ggagtccggc
     3121 tgatgttggg agtaggtggc tacgtctccg aactcacgac cgaaaagatc aagagcagcc
     3181 cgcatggatt tgacttggtc agggccgagc ctacatgtgc gaatgatgcc catacttgag
     3241 ccacctaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctgcgtaac
     3301 atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt gctgcttgga
     3361 tgcccgaggc atagactgta caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc
     3421 gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta
     3481 cttgcattac agtttacgaa ccgaacaggc ttatgtcaac tgggttcgtg ccttcatccg
     3541 tttccacggt gtgcgtcacc cggcaacctt gggcagcagc gaagtcgagg catttctgtc
     3601 ctggctggcg aacgagcgca aggtttcggt ctccacgcat cgtcaggcat tggcggcctt
     3661 gctgttcttc tacggcaagg tgctgtgcac ggatctgccc ttgcttcagg agatcggtag
     3721 acctcggccg tcgcggcgct tgccggtggt gctgaccccg gatgaagtgg ttcgcatcct
     3781 cggttttctg gaaggcgagc atcgtttgtt cgcccaggac tctagctata gttctagtgg
     3841 ttggctacgt acccgtagtg gctatggcag ggcttgccgc cccgacgttg gctgcgagcc
     3901 ctgggccttc acccgaactt gggggttggg gtggggaaaa ggaagaaacg cgggcgtatt
     3961 ggtcccaatg gggtctcggt ggggtatcga cagagtgcca gccctgggac cgaaccccgc
     4021 gtttatgaac aaacgaccca acacccgtgc gttttattct gtctttttat tgccgtcata
     4081 gcgcgggttc cttccggtat tgtctccttc cgtgtttcag ttagcctccc ccatctcccg
     4141 gtaccgcatg ctatgcatca gctgctagca ccatggctcg agatcccggg tgatcaagtc
     4201 ttcgtcgagt gattgtaaat aaaatgtaat ttacagtata gtattttaat taatatacaa
     4261 atgatttgat aataattctt atttaactat aatatattgt gttgggttga attaaaggtc
     4321 cgtatactcc ggaatattaa tagatcatgg agataattaa aatgataacc atctcgcaaa
     4381 taaataagta ttttactgtt ttcgtaacag ttttgtaata aaaaaaccta taaatattcc
     4441 ggattattca taccgtccca ccatcgggcg cggatccatg gcatccccta tactaggtta
     4501 ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa
     4561 atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga
     4621 attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca
     4681 gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa
     4741 agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc
     4801 gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc
     4861 tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca
     4921 tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc
     4981 aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc
     5041 acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca
     5101 agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggttccgc gtggatcagg
     5161 atcaggagag aatttatact ttcagggagc gcgcatggtg agcaagggcg aggagctgtt
     5221 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     5281 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     5341 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     5401 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     5461 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     5521 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     5581 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     5641 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     5701 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     5761 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag
     5821 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     5881 gatcactctc ggcatggacg agctgtacaa gggcggtgga ggcatgcaat ccacatccaa
     5941 ccacctctgg ctcctctcgg acattctcgg acaaggcgct accgctaacg tgttcagggg
     6001 aaggcacaag aagaccggtg acctgttcgc catcaaggtg ttcaacaaca tctcattcct
     6061 gcgccctgtg gacgtccaga tgcgtgagtt cgaagtgctg aagaagctga accacaagaa
     6121 catcgtcaag ctgttcgcca tcgaggaaga aaccactacc cgccacaagg tgctgatcat
     6181 ggagttctgc ccctgcggtt ccctgtacac tgtcctggaa gagccttcca acgcttacgg
     6241 tctgcccgag agcgagttcc tgatcgtcct gcgtgacgtg gtcggtggca tgaaccacct
     6301 gagggaaaac ggcatcgtgc acagagacat caagccagga aacatcatgc gcgtgatcgg
     6361 cgaggacgga cagagcgtct acaagctgac cgacttcggc gctgccagag agctggaaga
     6421 cgacgaacag ttcgtctctc tgtacggaac tgaagagtac ctgcaccctg acatgtacga
     6481 gagggccgtg ctgagaaagg accaccagaa gaagtacggt gctaccgtgg acctgtggtc
     6541 tatcggcgtc accttctacc acgctgccac tggatcactg cctttccgcc ccttcgaagg
     6601 tcctcgccgt aacaaggagg tcatgtacaa gatcatcact ggcaagccct ctggcgccat
     6661 ctcaggagtc cagaaggctg aaaacggtcc catcgactgg agcggcgaca tgccagtgag
     6721 ctgctctctg tcccgcggtc tgcaagtgct gctgacccca gtcctggcca acatcctgga
     6781 agctgaccag gagaagtgct ggggtttcga ccagttcttc gctgaaacct ccgacatcct
     6841 gcacagaatg gtcatccacg tcttcagcct gcaacagatg actgcccaca agatttacat
     6901 ccactcttac aacactgcta ctatcttcca cgaactggtc tacaagcaga ccaagatcat
     6961 ctccagcaac caggaactga tctacgaggg aaggagactg gtgctggagc ccggtcgcct
     7021 ggctcagcac ttcccaaaga ctaccgaaga gaaccctatc ttcgtggtgt cccgcgagcc
     7081 actgaacact atcggcctga tctacgagaa gatcagcctg cccaaggtcc acccacgtta
     7141 cgacctggac ggcgacgcta gcatggctaa ggccatcacc ggagtggtct gctacgcttg
     7201 ccgcatcgct tcaactctgc tgctgtacca ggaactgatg cgcaagggaa tccgttggct
     7261 gatcgagctg atcaaggacg actacaacga aactgtgcac aagaagaccg aggtggtcat
     7321 cactctggac ttctgcatcc gtaacatcga aaagaccgtg aaggtctacg agaagctgat
     7381 gaagatcaac ctggaggctg ccgaactggg agagatcagc gacatccaca ctaagctgct
     7441 gcgcctgtct tcatcccagg gcactatcga aacctccctg caagacatcg actcccgtct
     7501 gagcccagga ggttcactgg ctgacgcttg ggctcaccag gaaggcaccc accctaagga
     7561 caggaacgtg gagaagctgc aagtcctgct gaactgcatg actgaaatct actaccagtt
     7621 caagaaggac aaggccgagc gccgtctggc ttacaacgaa gagcagatcc acaagttcga
     7681 caagcagaag ctgtactacc acgccaccaa ggctatgact cacttcaccg acgaatgcgt
     7741 caagaagtac gaggctttcc tgaacaagag cgaagagtgg atcaggaaga tgctgcacct
     7801 gagaaagcag ctgctgtccc tgaccaacca gtgcttcgac atcgaagagg aagtgagcaa
     7861 gtaccaggag tacaccaacg agctgcaaga gactctgcct cagaagatgt tcactgccag
     7921 ctaaaagctt gtcgagaagt actagaggat cataatcagc cataccacat ttgtagaggt
     7981 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     8041 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     8101 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     8161 catcaatgta tcttatcatg tctggatctg atcactgctt gagcctagga gatccgaacc
     8221 agataagtga aatctagttc caaactattt tgtcattttt aattttcgta ttagcttacg
     8281 acgctacacc cagttcccat ctattttgtc actcttccct aaataatcct taaaaactcc
     8341 atttccaccc ctcccagttc ccaactattt tgtccgccca cagcggggca tttttcttcc
     8401 tgttatgttt ttaatcaaac atcctgccaa ctccatgtga caaaccgtca tcttcggcta
     8461 ctttttctct gtcacagaat gaaaattttt ctgtcatctc ttcgttatta atgtttgtaa
     8521 ttgactgaat atcaacgctt atttgcagcc tgaatggcga atgggacgcg ccctgtagcg
     8581 gcgcattaag cgcggcg
//
