LOCUS       sequence-430396-        6161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression in E. coli of NAP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6161)
  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 6161)
  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-NAP1
FEATURES             Location/Qualifiers
     source          1..6161
                     /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"
     primer_bind     complement(2211..2233)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2378..2396)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2464..2568
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2569..2637,2638..3429)
                     /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(2787..2806)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3600..4188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4089..4108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4342..4359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4432..4509
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4510..5592
                     /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(4529..4548)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5641..5646,5647..5664,5665..5671)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5679..5695
                     /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     5703..5719
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5703..5719
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5715..6161,1..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(5735..5752)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5735..5751)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5744..5766)
                     /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 agggaggagg aatggatgca ctggtagaag atgatatctg tattctgaat catgaaaaag
     1021 cccataagag agatacagtg actccagttt caatatattc aggagatgaa tctgttgctt
     1081 cccattttgc tcttgtcact gcatatgaag acatcaaaaa acgacttaag gattcagaga
     1141 aagagaactc tttgttaaag aagagaataa gatttttgga agaaaagcta atagctcgat
     1201 ttgaagaaga aacaagttcc gtgggacgag aacaagtaaa taaggcctat catgcatatc
     1261 gagaggtttg cattgataga gataatttga agagcaaact ggacaaaatg aataaagaca
     1321 actctgaatc tttgaaagta ttgaatgagc agctacaatc taaagaagta gaactcctcc
     1381 agctgaggac agaggtggaa actcagcagg tgatgaggaa tttaaatcca ccttcatcaa
     1441 actgggaggt ggaaaagttg agctgtgacc tgaagatcca tggtttggaa caagagctgg
     1501 aactgatgag gaaagaatgt agcgatctca aaatagaact acagaaagcc aaacaaacgg
     1561 atccatatca ggaagacaat ctgaagagca gagatctcca aaaactaagc atttcaagtg
     1621 ataatatgca gcatgcatac tgggaactga agagagaaat gtctaattta catctggtga
     1681 ctcaagtaca agctgaacta ctaagaaaac tgaaaacctc aactgcaatc aagaaagcct
     1741 gtgcccctgt aggatgcagt gaagaccttg gaagagacag cacaaaactg cacttgatga
     1801 attttactgc aacatacaca agacatcccc ctctcttacc aaatggcaaa gctctttgtc
     1861 ataccacatc ttccccttta ccaggagatg taaaggtttt atcagagaaa gcaatcctcc
     1921 aatcatggac agacaatgag agatccattc ctaatgatgg tacatgcttt caggaacaca
     1981 gttcttatgg cagaaattct ctggaagaca attcctgggt atttccaagt cctcctaaat
     2041 caagtgagac agcatttggg gaaactaaaa ctaaaacttt gcctttaccc aaccttccac
     2101 cactgcatta cttggatcaa cataatcaga actgccttta taagaattaa gcggccgcat
     2161 cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2221 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2281 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     2341 agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta
     2401 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     2461 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2521 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2581 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2641 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2701 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2761 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     2821 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2881 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2941 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3001 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3061 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     3121 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3181 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3241 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3301 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3361 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3421 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3481 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3541 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3601 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3661 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3721 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3781 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3841 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3901 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3961 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4021 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4081 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4141 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4201 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4261 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4321 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4381 cggtattttc tccttacgca tctgtgcggt atttcacacc gcataaattc cgacaccatc
     4441 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     4501 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     4561 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     4621 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     4681 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     4741 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     4801 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     4861 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     4921 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     4981 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     5041 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     5101 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     5161 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     5221 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     5281 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     5341 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     5401 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     5461 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     5521 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     5581 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccccaggc
     5641 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     5701 cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg
     5761 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     5821 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     5881 atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg
     5941 agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt
     6001 acgatgcgcc catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc
     6061 ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac
     6121 aggaaggcca gacgcgaatt atttttgatg gcgttggaat t
//
