LOCUS       sequence-448303-        7439 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 7439)
  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 7439)
  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: pETDuet1_BCL2L13(1-465aa)-THROMBIN-GST
            (W276A/...
FEATURES             Location/Qualifiers
     source          1..7439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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             1698..1715
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1722..2375
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2330..2352
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2388..2432
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2470..2488)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2484..2531
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2568..3023
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2655..2674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2865..2886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3138..3929,3930..3998))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3761..3780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3999..4091)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4172..4760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4661..4680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4914..4931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4946..5085
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5011..5030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5171..5193
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5187..5378)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5950..7032)
                     /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     6994..7013
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7033..7110)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7316..7335
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7423..7439,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tggcgtcctc ttctactgtg cctctgggat ttcactatga aacaaagtat gttgttctca
      361 gctacttggg actcctctct caagagaagc tgcaagagca acatctttcc tcaccccaag
      421 gggttcaact agatatagct tcacaatctc tggatcaaga aattttatta aaagttaaaa
      481 ctgaaattga agaagagcta aaatctctgg acaaagaaat ttctgaagcc ttcaccagca
      541 caggctttga ccgtcacact tctccagtgt tcagccctgc caatccagaa agctcaatgg
      601 aagactgctt ggcccatctt ggagaaaaag tgtcccagga actgaaagag cctctccata
      661 aagcattgca aatgctcctg agccagccag tgacatatca ggcatttcgg gaatgtacac
      721 tggagaccac agttcatgcc agcggctgga ataagatttt ggtgcctctg gttttgctac
      781 gacaaatgct tttggaattg acaagacgtg gtcaagaacc tttgagcgca ctgctgcagt
      841 ttggcgtgac atacctggag gactattcgg cagagtacat cattcagcaa ggtggctggg
      901 gcactgtgtt tagtcttgag tcagaggagg aggaataccc tggaatcact gcagaagata
      961 gcaatgacat ttacatcctg cccagcgaca actctggaca agtcagtccc ccagagtctc
     1021 caactgtgac cacttcctgg cagtctgaga gcttacctgt gtcactgtca gctagccaga
     1081 gttggcacac agaaagcctg ccagtgtcac taggccctga gtccgcgcag caggctgcaa
     1141 tggatcctga agaagtgaaa agcttagaca gcaacggagc tggagagaag agtgagaaca
     1201 actcctctaa ttctgacgct gtgcacgcgg agaaagaaga ggtgcccgag ggcatggaag
     1261 aggctgctgt ggcttctgtg gtcttgccag cgcgggagct gcaagaggca cttcctgaag
     1321 ccccagctcc cttgcttcca catatcactg ccacctccct gctggggaca agggaacctg
     1381 acacagaagt gatcacagtt gagaaatcca gccctgctac atctctgttt gtagaacttg
     1441 atgaagaaga ggtgaaagca gcaacaactg aacctactga agtggaggag gtggtccccg
     1501 cactggaacc cacagaaacg ctgctgagtg agaaggagat aaacgcaagg gaagagagcc
     1561 ttgtggaaga gctgtcccct gccagcgaga agaagcccgt gccgccgtct gagggcaagt
     1621 ctagactgtc ccccgccggt gagatgaagc ccatgccgct gtctgagggc aagtctatac
     1681 tgctgtttgg aggatcactg gttccgcgtg gatccggcgg atcccctata ctaggttatt
     1741 ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat
     1801 atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat
     1861 tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt
     1921 ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag
     1981 agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga
     2041 gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg
     2101 aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg
     2161 taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa
     2221 tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac
     2281 aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag
     2341 ccacgtttgg tggtggcgac catcctccaa aatagctcga gtctggtaaa gaaaccgctg
     2401 ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg
     2461 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2521 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     2581 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2641 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2701 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2761 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2821 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2881 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2941 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3001 caaaatatta acgtttacaa tttctggcgg cacgatggca tgagattatc aaaaaggatc
     3061 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3121 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3181 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3241 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3301 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3361 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3421 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3481 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3541 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3601 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3661 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3721 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3781 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3841 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3901 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3961 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatcatga
     4021 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4081 aaataaacaa ataggtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4141 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4201 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4261 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4321 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4381 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4441 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4501 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4561 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4621 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4681 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4741 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4801 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4861 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4921 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4981 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5041 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     5101 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5161 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5221 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5281 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5341 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5401 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5461 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcgatatgg
     5521 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5581 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5641 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5701 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5761 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5821 aggagcacga tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5881 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5941 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6001 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     6061 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     6121 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6181 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     6241 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6301 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     6361 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6421 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6481 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6541 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6601 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6661 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6721 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6781 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6841 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6901 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6961 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     7021 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     7081 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     7141 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     7201 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     7261 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     7321 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     7381 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactata
//
