LOCUS       sequence-447444-        5320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant protein for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5320)
  AUTHORS   Adriaenssens E, Schaar S, Cook ASI, Stuke JFM, Sawa-Makarska J, 
            Nguyen TN, Ren X, Schuschnig M, Romanov J, Khuu G, Uoselis L, 
            Lazarou M, Hummer G, Hurley JH, Martens S
  TITLE     Reconstitution of BNIP3/NIX-mitophagy initiation reveals 
            hierarchical flexibility of the autophagy machinery.
  JOURNAL   Nat Cell Biol. 2025 Jul 25. doi: 10.1038/s41556-025-01712-y.
  PUBMED    40715440
REFERENCE   2  (bases 1 to 5320)
  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: "Nat Cell 
            Biol. 2025 Jul 25. doi: 10.1038/s41556-025-01712-y."
COMMENT     Sequence Label: pGEX-4T1_GST-LC3A
FEATURES             Location/Qualifiers
     source          1..5320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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             256..909
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     867..889
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             916..933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             943..1299
                     /codon_start=1
                     /gene="MAP1LC3A"
                     /product="human LC3 protein"
                     /label=LC3A
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSDRPFKQRRSFADRCKEVQQIRDQHPSKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELVKIIRRRLQLNPTQAFFLLVNQHSMVSVSTPIADIYEQEKDE
                     DGFLYMVYASQETFG"
     RBS             990..998
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(1368..1390)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1535..1553)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1621..1725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1726..1794,1795..2586)
                     /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(1944..1963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2757..3345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3246..3265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3499..3516
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3589..3666
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3667..4749
                     /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(3686..3705)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4798..4803,4804..4821,4822..4828)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4836..4852
                     /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     4860..4876
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4860..4876
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4872..5320,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4892..4909)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4892..4908)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4901..4923)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      301 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      361 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      421 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      481 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      541 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      601 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      661 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      721 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      781 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      841 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      901 cctccaaaat cggatctggt tccgcgtgga tccccggaat tcccctcaga ccggcctttc
      961 aagcagcggc ggagcttcgc cgaccgctgt aaggaggtac agcagatccg cgaccagcac
     1021 cccagcaaaa tcccggtgat catcgagcgc tacaagggtg agaagcagct gcccgtcctg
     1081 gacaagacca agtttttggt cccggaccat gtcaacatga gcgagttggt caagatcatc
     1141 cggcgccgcc tgcagctgaa ccccacgcag gccttcttcc tgctggtgaa ccagcacagc
     1201 atggtgagtg tgtccacgcc catcgcggac atctacgagc aggagaaaga cgaggacggc
     1261 ttcctctata tggtctacgc ctcccaggaa accttcggct gactcgagcg gccgcatcgt
     1321 gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     1381 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     1441 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     1501 gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     1561 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     1621 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1681 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1741 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1801 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1861 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1921 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     1981 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2041 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2101 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2161 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2221 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     2281 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2341 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2401 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2461 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2521 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2581 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2641 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2701 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2761 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2821 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2881 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2941 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3001 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3061 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3121 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3181 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3241 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3301 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3361 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3421 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3481 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3541 tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa
     3601 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     3661 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     3721 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     3781 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     3841 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     3901 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     3961 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     4021 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     4081 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     4141 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     4201 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     4261 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     4321 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     4381 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     4441 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     4501 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     4561 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     4621 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     4681 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     4741 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt
     4801 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4861 aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact
     4921 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     4981 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     5041 gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt
     5101 gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg
     5161 atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca
     5221 cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg
     5281 aaggccagac gcgaattatt tttgatggcg ttggaattag
//
