LOCUS       sequence-448034-        6571 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 6571)
  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 6571)
  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: pCAG_GST-TEV-WIPI3
FEATURES             Location/Qualifiers
     source          1..6571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(163..179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(163..179)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(176..198)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    187..203
                     /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)."
     promoter        complement(join(211..217,218..235,236..241))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    256..277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        336..531
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      382..517
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    537..671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(574..593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     628..647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(739..756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(910..1498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(990..1009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1669..2460,2461..2529))
                     /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     2292..2311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2530..2634)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2665..3044
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3046..3321
                     /label=chicken beta-actin promoter
     intron          3322..4338
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4262..4284
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4346..4365
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4412..4421
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4418..5074
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     5032..5054
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             5102..5122
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(6253..6272)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6318..6373
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6372..6391)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
       61 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
      121 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
      181 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      241 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
      301 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
      361 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      421 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      481 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
      541 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      601 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
      661 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
      721 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
      781 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
      841 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
      901 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
      961 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1021 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1081 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1141 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1201 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1261 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1321 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     1381 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     1441 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     1501 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     1561 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     1621 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     1681 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     1741 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     1801 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     1861 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     1921 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     1981 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     2041 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     2101 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     2161 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     2221 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     2281 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     2341 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     2401 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     2461 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     2521 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     2581 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     2641 tccccgaaaa gtgccacctg ggtcgacatt gattattgac tagttattaa tagtaatcaa
     2701 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2761 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2821 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2881 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2941 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     3001 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     3061 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     3121 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gccaggcggg
     3181 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
     3241 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     3301 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     3361 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     3421 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
     3481 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
     3541 agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg
     3601 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg
     3661 cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac
     3721 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc
     3781 gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt
     3841 gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag
     3901 gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc
     3961 ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
     4021 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
     4081 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
     4141 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
     4201 ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
     4261 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     4321 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     4381 ggcaaagaat tcctcgatcg acggtatcga tgccaccatg gcatccccta tactaggtta
     4441 ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa
     4501 atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga
     4561 attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca
     4621 gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa
     4681 agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc
     4741 gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc
     4801 tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca
     4861 tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc
     4921 aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc
     4981 acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca
     5041 agccacgttt ggtggtggcg accatcctcc aaaatcggat gattacgata tcccaacgac
     5101 cgaaaacctg tattttcagg gtggtaccat gaatctgctg ccttgtaatc cacacgggaa
     5161 cgggctgctg tatgctgggt ttaatcagga ccacggctgc ttcgcttgcg gaatggagaa
     5221 cgggttcaga gtgtacaata ccgacccact gaaagagaag gaaaaacagg agttcctgga
     5281 aggcggggtc ggacatgtgg aaatgctgtt taggtgcaac tacctggctc tggtgggagg
     5341 cgggaagaaa cccaagtatc cccctaacaa agtgatgatc tgggacgatc tgaagaaaaa
     5401 gactgtgatc gagattgagt tcagcaccga ggtcaaggcc gtgaaactgc ggagagaccg
     5461 catcgtggtc gtgctggatt ccatgatcaa ggtcttcacc tttacacaca accctcatca
     5521 gctgcacgtg ttcgaaacat gctataaccc caagggcctg tgcgtgctgt gccctaattc
     5581 aaacaatagc ctgctggctt ttcccggaac tcataccggc cacgtccagc tggtggacct
     5641 ggcatctact gagaagccac ccgtcgatat ccctgcacat gaaggggtgc tgtcctgtat
     5701 cgccctgaat ctgcagggaa cacgaattgc tactgcatct gagaagggca ccctgatccg
     5761 gattttcgac acaagctccg gccacctgat tcaggaactg aggcgcggga gtcaggccgc
     5821 taacatctac tgcattaact tcaatcagga cgccagcctg atctgcgtga gcagtgatca
     5881 tggcaccgtg cacatttttg cagccgagga tccaaagcga aacaagcagt caagcctggc
     5941 cagtgcttca ttcctgccca agtatttttc ctctaaatgg agcttctcca agtttcaggt
     6001 gccttctggg agtccatgca tctgtgcctt tggaacagag cctaacgcag tgatcgccat
     6061 ttgtgctgat ggcagctact ataaattcct gtttaatcca aagggggagt gtattaggga
     6121 tgtctatgct cagttcctgg agatgaccga cgataagctg taactcgagg aattcactcc
     6181 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     6241 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     6301 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     6361 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     6421 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg
     6481 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     6541 aaaagccttg acttgaggtt agattttttt t
//
