LOCUS       sequence-455265-        6998 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 6998)
  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 6998)
  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: pETDuet_6xHis-TEV-mCherry-ATG13 IDR(230-517aa)
FEATURES             Location/Qualifiers
     source          1..6998
                     /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"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             134..838
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(276..294)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     544..563
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1795..1813
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1814..1838
                     /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             1947..1991
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2029..2047)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2043..2090
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2127..2582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2214..2233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2424..2445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2697..3488,3489..3557))
                     /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     3320..3339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3558..3650)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3731..4319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4220..4239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4473..4490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4505..4644
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4570..4589)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4730..4752
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4746..4937)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5509..6591)
                     /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     6553..6572
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6592..6669)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6875..6894
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6982..6998,1..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 cggaaaacct
      121 gtattttcag ggagtgagca agggcgagga ggataacatg gccatcatca aggagttcat
      181 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
      241 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      301 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
      361 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
      421 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
      481 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
      541 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
      601 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
      661 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
      721 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
      781 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
      841 cggactcaga tctcgagctc aagcttcgaa ctatcgtaca gccggtgaag acaccggcgt
      901 gatctacccc agtgtcgagg atagccagga agtgtgtacc acttccttct ctacctcacc
      961 accctcacag ctctcttcaa gtcgcctgag ttatcagcct gccgctctcg gagtcggtag
     1021 cgcagacctg gcctacccag tggtcttcgc agcaggcctc aacgcaaccc acccacatca
     1081 gctgatggtg ccaggaaagg agggtggagt cccactggct cctaatcagc cagtgcacgg
     1141 tactcaggct gaccaggaaa ggctggcaac ttgcacacca tccgatagaa ctcattgtgc
     1201 tgcaacaccc agctcctctg aggacaccga aactgtgagc aactcaagtg agggtcgcgc
     1261 ctccccacac gatgtcctgg aaaccatctt cgtgagaaaa gtcggcgctt ttgtgaacaa
     1321 gcccattaat caggtcacac tcacctctct ggacatccct ttcgctatgt ttgcaccaaa
     1381 gaacctcgag ctggaagaca ctgatcccat ggtgaatcct ccagactcac ccgagacaga
     1441 aagtcctctc cagggcagtc tgcacagcga tggaagctcc ggtggctctt caggcaacac
     1501 acatgacgat ttcgtcatga ttgacttcaa acccgctttt tccaaggacg atatcctccc
     1561 tatggatctg ggaaccttct acagggagtt tcagaatccc cctcagctca gtagcctgag
     1621 cattgacatc ggtgcccagt ccatggctga ggacctcgat tctctgcctg aaaaactggc
     1681 tgtgcacgag aagaacgtca gagaattcga cgcatttgtg gagaccctgc agtgagcata
     1741 atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg aaattaatac
     1801 gactcactat aggggaattg tgagcggata acaattcccc atcttagtat attagttaag
     1861 tataagaagg agatatacat atggcagatc tcaattggat atcggccggc cacgcgatcg
     1921 ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc
     1981 acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat
     2041 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2101 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2161 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2221 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2281 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2341 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2401 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2461 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2521 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     2581 ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2641 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2701 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2761 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2821 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2881 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2941 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3001 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3061 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3121 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3181 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3241 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3301 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3361 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3421 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3481 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3541 aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt atcagggtta
     3601 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggtcatga
     3661 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3721 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3781 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3841 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3901 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3961 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4021 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4081 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4141 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4201 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4261 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4321 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4381 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4441 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4501 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4561 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4621 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4681 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4741 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4801 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4861 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4921 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4981 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     5041 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     5101 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5161 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     5221 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5281 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5341 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc
     5401 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5461 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5521 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5581 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5641 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5701 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5761 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     5821 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5881 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5941 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6001 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6061 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6121 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6181 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6241 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6301 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6361 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6421 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6481 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6541 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6601 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6661 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6721 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6781 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6841 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     6901 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     6961 tcgatctcga tcccgcgaaa ttaatacgac tcactata
//
