LOCUS       sequence-418255-        6884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression in E. coli of GFP-NAP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6884)
  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 6884)
  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: pGEX-4T1-GST-TEV-GFP-NAP1
FEATURES             Location/Qualifiers
     source          1..6884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             945..965
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             969..1682
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2934..2956)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3101..3119)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3187..3291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3292..3360,3361..4152)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3510..3529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4323..4911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4812..4831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5065..5082
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5155..5232
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5233..6315
                     /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     complement(5252..5271)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6364..6369,6370..6387,6388..6394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6402..6418
                     /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)."
     primer_bind     6426..6442
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6426..6442
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6438..6884,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6458..6475)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6458..6474)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6467..6489)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccccgga attcgaaaac ctgtattttc
      961 agggaggaat gagcaagggc gaggaactgt tcaccggtgt ggttccgatt ctggtggaac
     1021 tggacggcga tgttaacggt cacaaattta gcgttagcgg cgagggcgaa ggtgatgcga
     1081 cctatggcaa gctgaccctg aaattcatct gcaccaccgg taaactgccg gtgccgtggc
     1141 cgaccctggt taccaccctg acctacggtg ttcagtgctt tagccgttat ccggaccaca
     1201 tgaagcaaca cgatttcttt aaaagcgcga tgccggaggg ctacgtgcag gaacgtacca
     1261 tcttctttaa ggacgatggt aactataaaa cccgtgcgga agtgaagttc gaaggcgaca
     1321 ccctggttaa ccgtatcgag ctgaagggta ttgactttaa agaagatggc aacattctgg
     1381 gtcacaagct ggagtacaac tataacagcc acaacgtgta catcatggcg gataagcaga
     1441 aaaacggcat taaggttaac ttcaaaatcc gtcacaacat tgaagacggt agcgtgcaac
     1501 tggcggatca ctaccaacag aacaccccga ttggcgacgg tccggttctg ctgccggata
     1561 accactatct gagcacccag agcgcgctga gcaaggaccc gaacgaaaaa cgtgatcaca
     1621 tggtgctgct ggagtttgtg accgcggcgg gcattaccca cggcatggac gagctgtata
     1681 aaggtggtgg tggtatggat gcactggtag aagatgatat ctgtattctg aatcatgaaa
     1741 aagcccataa gagagataca gtgactccag tttcaatata ttcaggagat gaatctgttg
     1801 cttcccattt tgctcttgtc actgcatatg aagacatcaa aaaacgactt aaggattcag
     1861 agaaagagaa ctctttgtta aagaagagaa taagattttt ggaagaaaag ctaatagctc
     1921 gatttgaaga agaaacaagt tccgtgggac gagaacaagt aaataaggcc tatcatgcat
     1981 atcgagaggt ttgcattgat agagataatt tgaagagcaa actggacaaa atgaataaag
     2041 acaactctga atctttgaaa gtattgaatg agcagctaca atctaaagaa gtagaactcc
     2101 tccagctgag gacagaggtg gaaactcagc aggtgatgag gaatttaaat ccaccttcat
     2161 caaactggga ggtggaaaag ttgagctgtg acctgaagat ccatggtttg gaacaagagc
     2221 tggaactgat gaggaaagaa tgtagcgatc tcaaaataga actacagaaa gccaaacaaa
     2281 cggatccata tcaggaagac aatctgaaga gcagagatct ccaaaaacta agcatttcaa
     2341 gtgataatat gcagcatgca tactgggaac tgaagagaga aatgtctaat ttacatctgg
     2401 tgactcaagt acaagctgaa ctactaagaa aactgaaaac ctcaactgca atcaagaaag
     2461 cctgtgcccc tgtaggatgc agtgaagacc ttggaagaga cagcacaaaa ctgcacttga
     2521 tgaattttac tgcaacatac acaagacatc cccctctctt accaaatggc aaagctcttt
     2581 gtcataccac atcttcccct ttaccaggag atgtaaaggt tttatcagag aaagcaatcc
     2641 tccaatcatg gacagacaat gagagatcca ttcctaatga tggtacatgc tttcaggaac
     2701 acagttctta tggcagaaat tctctggaag acaattcctg ggtatttcca agtcctccta
     2761 aatcaagtga gacagcattt ggggaaacta aaactaaaac tttgccttta cccaaccttc
     2821 caccactgca ttacttggat caacataatc agaactgcct ttataagaat taagcggccg
     2881 catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     2941 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3001 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     3061 cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt
     3121 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3181 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3241 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3301 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3361 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3421 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3481 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     3541 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3601 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3661 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3721 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3781 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     3841 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3901 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3961 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4021 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4081 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4141 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4201 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4261 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4321 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4381 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4441 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4501 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4561 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4621 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4681 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4741 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4801 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4861 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4921 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4981 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5041 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     5101 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc
     5161 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     5221 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     5281 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     5341 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     5401 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     5461 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     5521 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     5581 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     5641 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     5701 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     5761 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     5821 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     5881 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     5941 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     6001 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     6061 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     6121 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     6181 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     6241 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     6301 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca
     6361 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6421 tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc
     6481 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     6541 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     6601 tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc
     6661 tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg
     6721 gttacgatgc gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg
     6781 ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc
     6841 tacaggaagg ccagacgcga attatttttg atggcgttgg aatt
//
