LOCUS       sequence-455261-        6741 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 6741)
  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 6741)
  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-CCPG1(1-212aa)-thrombin-GST
FEATURES             Location/Qualifiers
     source          1..6741
                     /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             755..772
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             779..1432
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1387..1409
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        1535..1553
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1554..1578
                     /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             1687..1731
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1769..1787)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1783..1830
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1867..2322
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1954..1973)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2164..2185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2437..3228,3229..3297))
                     /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     3060..3079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3298..3390)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3471..4059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3960..3979
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4213..4230
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4245..4387
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4313..4332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4473..4495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4489..4680)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5252..6334)
                     /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     6296..6315
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6335..6412)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6618..6637
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6725..6741,1..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 caatgtctga
      121 aaattccagt gacagtgatt catcttgtgg ttggactgtc atcagtcatg aggggtcaga
      181 tatagaaatg ttgaattctg tgacccccac tgacagctgt gagcccgccc cagaatgttc
      241 atctttagag caagaggagc ttcaagcatt gcagatagag caaggagaaa gcagccaaaa
      301 tggcacagtg cttatggaag aaactgctta tccagctttg gaggaaacca gctcaacaat
      361 tgaggcagag gaacaaaaga tacccgaaga cagtatctat attggaactg ccagtgatga
      421 ttctgatatt gttacccttg agccacctaa gttagaagaa attggaaatc aagaagttgt
      481 cattgttgaa gaagcacaga gttcagaaga ctttaacatg ggctcttcct ctagcagcca
      541 gtatactttc tgtcagccag aaactgtatt ttcatctcag cctagtgacg atgaatcaag
      601 tagtgatgaa accagtaatc agcccagtcc tgcctttaga cgacgccgtg ctaggaagaa
      661 gaccgtttct gcttcagaat ctgaagaccg gctagttgct gaacaagaaa ctgaaccttc
      721 taaggagttg agtaaacgtc agttcagtgg atcactggtt ccgcgtggat ccggcggatc
      781 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      841 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      901 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      961 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
     1021 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
     1081 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
     1141 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
     1201 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
     1261 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
     1321 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
     1381 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat aggaattcga
     1441 gctcggcgcg cctgcaggtc gacaagcttg cggccgcata atgcttaagt cgaacagaaa
     1501 gtaatcgtat tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg
     1561 tgagcggata acaattcccc atcttagtat attagttaag tataagaagg agatatacat
     1621 atggcagatc tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag
     1681 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
     1741 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     1801 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1861 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1921 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1981 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2041 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2101 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2161 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2221 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2281 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat
     2341 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2401 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2461 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2521 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2581 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2641 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2701 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2761 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2821 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2881 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2941 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3001 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3061 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3121 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3181 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3241 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3301 cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3361 tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag
     3421 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3481 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3541 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3601 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3661 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3721 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3781 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3841 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3901 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3961 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4021 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4081 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4141 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4201 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4261 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     4321 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4381 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4441 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4501 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4561 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4621 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4681 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4741 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4801 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4861 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4921 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4981 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5041 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5101 gcctagccgg gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg
     5161 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5221 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5281 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5341 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5401 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5461 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5521 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5581 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5641 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5701 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5761 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5821 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5881 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5941 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     6001 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6061 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6121 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6181 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6241 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6301 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6361 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6421 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6481 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6541 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6601 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6661 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     6721 aaattaatac gactcactat a
//
