LOCUS       sequence-455256-        5620 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of recombinant protein for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5620)
  AUTHORS   Adriaenssens E, Schaar S, Cook ASI, Stuke JFM, Sawa-Makarska J, 
            Nguyen TN, Ren X, Schuschnig M, Romanov J, Khuu G, Lazarou M, Hummer
            G, Hurley JH, Martens S
  TITLE     Reconstitution of BNIP3/NIX-mitophagy initiation reveals 
            hierarchical flexibility of the autophagy machinery
  JOURNAL   bioRxiv 2024.08.28.609967
REFERENCE   2  (bases 1 to 5620)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.08.28.609967"
COMMENT     Sequence Label: pGEX-4T1_GST-thrombin-FAM134C(250-466aa)
FEATURES             Location/Qualifiers
     source          1..5620
                     /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"
     primer_bind     complement(1670..1692)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1837..1855)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1923..2027
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2028..2096,2097..2888)
                     /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(2246..2265)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3059..3647
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3548..3567
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3801..3818
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3891..3968
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3969..5051
                     /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(3988..4007)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5064..5085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5100..5105,5106..5123,5124..5130)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5138..5154
                     /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     5162..5178
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5162..5178
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5174..5620,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5194..5211)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5194..5210)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5203..5225)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga 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 gatccccaga acgagccatg gacaaccaca
      961 gtgacagcga agaggagctt gctgccttct gtcctcagct ggacgattct actgttgcca
     1021 gggaattggc catcacagac tctgagcact cagacgctga agtctcctgt acagacaatg
     1081 gcacattcaa tctttcaagg ggccaaacac ctctaacgga aggctctgaa gacctagatg
     1141 gtcacagtga tccagaggaa tcctttgcca gagaccttcc agacttccct tccattaata
     1201 tggatcctgc tggcctggat gatgaggacg acactagcat tggcatgccc agcttgatgt
     1261 accgttctcc gccaggggct gaggagcccc aggccccacc tgccagccgg gacgaggctg
     1321 cgctgccgga gctcctgctt ggtgctcttc ctgtaggatc caacctcacc agcaaccttg
     1381 ccagcctggt ctcccagggt atgattcagc tggccttgtc aggggcctcc caaccaggcc
     1441 cttctggagc acctgcccag agagcaacga gaggcttcct ccggtccccc agttcagacc
     1501 tggacactga tgctgagggg gatgactttg aacttctgga ccagtcggag ctgagtcagc
     1561 tggaccctgc cagttctagg agccactgag aattcccggg tcgactcgag cggccgcatc
     1621 gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1681 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1741 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     1801 gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     1861 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     1921 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1981 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2041 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2101 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2161 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2221 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     2281 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2341 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2401 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2461 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2521 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2581 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2641 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2701 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2761 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2821 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2881 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2941 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3001 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3061 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3121 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3181 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3241 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3301 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3361 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3421 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3481 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3541 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3601 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3661 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3721 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3781 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3841 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg
     3901 aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     3961 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     4021 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     4081 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     4141 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     4201 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     4261 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     4321 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     4381 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     4441 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     4501 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     4561 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     4621 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     4681 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     4741 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     4801 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     4861 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     4921 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     4981 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5041 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     5101 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5161 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
     5221 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5281 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5341 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
     5401 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
     5461 cgatgcgccc atctacacca acgtaaccta tcccattacg gtcaatccgc cgtttgttcc
     5521 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
     5581 ggaaggccag acgcgaatta tttttgatgg cgttggaatt
//
