LOCUS       sequence-423068-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression in E. coli of NAP1 (delta-TBK1) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  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 6616)
  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-MBP-TEV-NAP1_delta-TBK1 (L226Q/L233Q)
FEATURES             Location/Qualifiers
     source          1..6616
                     /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..1358
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1332..1355
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1383..1403
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2666..2688)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2833..2851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2919..3023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3024..3092,3093..3884)
                     /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(3242..3261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4055..4643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4544..4563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4797..4814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4887..4964
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4965..6047
                     /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(4984..5003)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6096..6101,6102..6119,6120..6126)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6134..6150
                     /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     6158..6174
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6158..6174
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6170..6616,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTVRCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6190..6207)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6190..6206)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6199..6221)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgtacggtg 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 aaaatcgaag aaggtaaact ggtaatctgg attaacggcg
      301 ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta
      361 aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg
      421 gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
      481 gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct
      541 gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat
      601 cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg
      661 cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac
      721 cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg
      781 gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct
      841 tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag
      901 aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca
      961 acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac
     1021 catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag
     1081 agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta
     1141 ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag
     1201 atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc
     1261 cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc
     1321 gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg gctccggaat
     1381 tcgaaaacct gtattttcag ggaggaggaa tggatgcact ggtagaagat gatatctgta
     1441 ttctgaatca tgaaaaagcc cataagagag atacagtgac tccagtttca atatattcag
     1501 gagatgaatc tgttgcttcc cattttgctc ttgtcactgc atatgaagac atcaaaaaac
     1561 gacttaagga ttcagagaaa gagaactctt tgttaaagaa gagaataaga tttttggaag
     1621 aaaagctaat agctcgattt gaagaagaaa caagttccgt gggacgagaa caagtaaata
     1681 aggcctatca tgcatatcga gaggtttgca ttgatagaga taatttgaag agcaaactgg
     1741 acaaaatgaa taaagacaac tctgaatctt tgaaagtatt gaatgagcag ctacaatcta
     1801 aagaagtaga actcctccag ctgaggacag aggtggaaac tcagcaggtg atgaggaatt
     1861 taaatccacc ttcatcaaac tgggaggtgg aaaagttgag ctgtgacctg aagatccatg
     1921 gtttggaaca agagctggaa ctgatgagga aagaatgtag cgatctcaaa atagaactac
     1981 agaaagccaa acaaacggat ccatatcagg aagacaatct gaagagcaga gatctccaaa
     2041 aactaagcat ttcaagtgat aatatgcagc atgcatactg ggaacagaag agagaaatgt
     2101 ctaatcaaca tctggtgact caagtacaag ctgaactact aagaaaactg aaaacctcaa
     2161 ctgcaatcaa gaaagcctgt gcccctgtag gatgcagtga agaccttgga agagacagca
     2221 caaaactgca cttgatgaat tttactgcaa catacacaag acatccccct ctcttaccaa
     2281 atggcaaagc tctttgtcat accacatctt cccctttacc aggagatgta aaggttttat
     2341 cagagaaagc aatcctccaa tcatggacag acaatgagag atccattcct aatgatggta
     2401 catgctttca ggaacacagt tcttatggca gaaattctct ggaagacaat tcctgggtat
     2461 ttccaagtcc tcctaaatca agtgagacag catttgggga aactaaaact aaaactttgc
     2521 ctttacccaa ccttccacca ctgcattact tggatcaaca taatcagaac tgcctttata
     2581 agaattaatg acccgggtga ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg
     2641 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2701 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2761 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     2821 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2881 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2941 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3001 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3061 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3121 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3181 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3241 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3301 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3361 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3421 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3481 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3541 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3601 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3661 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3721 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3781 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3841 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3901 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3961 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4021 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4081 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4141 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4201 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4261 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4321 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4381 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4441 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4501 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4561 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4621 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4681 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4741 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4801 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4861 gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta
     4921 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4981 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     5041 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5101 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5161 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5221 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5281 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5341 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5401 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5461 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5521 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5581 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5641 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5701 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5761 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5821 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5881 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5941 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6001 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6061 aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6121 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     6181 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6241 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6301 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     6361 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     6421 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     6481 gacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     6541 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     6601 tgatggcgtt ggaatt
//
