LOCUS       sequence-376963-        7868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression of His-TEV-mCherry-OPTN WT. Internal 
            reference: SMC797.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7868)
  AUTHORS   Zaffagnini G, Savova A, Danieli A, Romanov J, Tremel S, Ebner M, 
            Peterbauer T, Sztacho M, Trapannone R, Tarafder AK, Sachse C, 
            Martens S
  TITLE     p62 filaments capture and present ubiquitinated cargos for 
            autophagy.
  JOURNAL   EMBO J. 2018 Mar 1;37(5). pii: embj.201798308. doi: 
            10.15252/embj.201798308. Epub 2018 Jan 17.
  PUBMED    29343546
REFERENCE   2  (bases 1 to 7868)
  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: "EMBO J."; 
            date: "2018-03-1"; volume: "37(5). pii: embj.201798308. doi"; pages:
            " 10.15252/embj.201798308. Epub 2018 Jan 17"
COMMENT     Sequence Label: pET-Duet1-His-TEV-mCherry-OPTN WT
FEATURES             Location/Qualifiers
     source          1..7868
                     /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             119..139
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             140..844
                     /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(282..300)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     550..569
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1183..1194
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        2665..2683
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2684..2708
                     /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             2817..2861
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2899..2917)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2913..2960
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2997..3452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3084..3103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3294..3315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3567..4358,4359..4427))
                     /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     4190..4209
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4428..4520)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4601..5189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5090..5109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5343..5360
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5375..5514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5440..5459)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5600..5622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5616..5807)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6379..7461)
                     /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     7423..7442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7462..7539)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7745..7764
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7852..7868,1..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 cgaattcgga
      121 aaacctgtat tttcagggag tgagcaaggg cgaggaggat aacatggcca tcatcaagga
      181 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
      241 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
      301 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
      361 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
      421 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
      481 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
      541 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
      601 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
      661 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
      721 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
      781 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
      841 caagtccgga ctcagatctc gagctatgtc ccatcaacct ctcagctgcc tcactgaaaa
      901 ggaggacagc cccagtgaaa gcacaggaaa tggacccccc cacctggccc acccaaacct
      961 ggacacgttt accccggagg agctgctgca gcagatgaaa gagctcctga ccgagaacca
     1021 ccagctgaaa gaagccatga agctaaataa tcaagccatg aaagggagat ttgaggagct
     1081 ttcggcctgg acagagaaac agaaggaaga acgccagttt tttgagatac agagcaaaga
     1141 agcaaaagag cgtctaatgg ccttgagtca tgagaatgag aaattgaagg aagagcttgg
     1201 aaaactaaaa gggaaatcag aaaggtcatc tgaggacccc actgatgact ccaggcttcc
     1261 cagggccgaa gcggagcagg aaaaggacca gctcaggacc caggtggtga ggctacaagc
     1321 agagaaggca gacctgttgg gcatcgtgtc tgaactgcag ctcaagctga actccagcgg
     1381 ctcctcagaa gattcctttg ttgaaattag gatggctgaa ggagaagcag aagggtcagt
     1441 aaaagaaatc aagcatagtc ctgggcccac gagaacagtc tccactggca cggcattgtc
     1501 taaatatagg agcagatctg cagatggggc caagaattac ttcgaacatg aggagttaac
     1561 tgtgagccag ctcctgctgt gcctaaggga agggaatcag aaggtggaga gacttgaagt
     1621 tgcactcaag gaggccaaag aaagagtttc agattttgaa aagaaaacaa gtaatcgttc
     1681 tgagattgaa acccagacag aggggagcac agagaaagag aatgatgaag agaaaggccc
     1741 ggagactgtt ggaagcgaag tggaagcact gaacctccag gtgacatctc tgtttaagga
     1801 gcttcaagag gctcatacaa aactcagcga agctgagcta atgaagaaga gacttcaaga
     1861 aaagtgtcag gcccttgaaa ggaaaaattc tgcaattcca tcagagttga atgaaaagca
     1921 agagcttgtt tatactaaca aaaagttaga gctacaagtg gaaagcatgc tatcagaaat
     1981 caaaatggaa caggctaaaa cagaggatga aaagtccaaa ttaactgtgc tacagatgac
     2041 acacaacaag cttcttcaag aacataataa tgcattgaaa acaattgagg aactaacaag
     2101 aaaagagtca gaaaaagtgg acagggcagt gctgaaggaa ctgagtgaaa aactggaact
     2161 ggcagagaag gctctggctt ccaaacagct gcaaatggat gaaatgaagc aaaccattgc
     2221 caagcaggaa gaggacctgg aaaccatgac catcctcagg gctcagatgg aagtttactg
     2281 ttctgatttt catgctgaaa gagcagcgag agagaaaatt catgaggaaa aggagcaact
     2341 ggcattgcag ctggcagttc tgctgaaaga gaatgatgct ttcgaagacg gaggcaggca
     2401 gtccttgatg gagatgcaga gtcgtcatgg ggcgagaaca agtgactctg accagcaggc
     2461 ttaccttgtt caaagaggag ctgaggacag ggactggcgg caacagcgga atattccgat
     2521 tcattcctgc cccaagtgtg gagaggttct gcctgacata gacacgttac agattcacgt
     2581 gatggattgc atcatttaag cggccgcata atgcttaagt cgaacagaaa gtaatcgtat
     2641 tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata
     2701 acaattcccc atcttagtat attagttaag tataagaagg agatatacat atggcagatc
     2761 tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag
     2821 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     2881 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2941 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     3001 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3061 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3121 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3181 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3241 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3301 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3361 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3421 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     3481 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3541 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3601 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3661 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3721 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3781 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3841 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3901 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3961 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4021 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4081 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4141 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4201 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4261 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4321 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4381 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4441 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4501 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4561 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4621 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4681 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4741 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4801 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4861 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4921 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4981 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5041 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5101 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5161 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5221 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5281 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5341 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     5401 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5461 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5521 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5581 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5641 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5701 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5761 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5821 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5881 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5941 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6001 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6061 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     6121 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6181 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6241 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     6301 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     6361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6421 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6481 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6541 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6601 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6661 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6721 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6781 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6841 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6901 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6961 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     7021 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     7081 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     7141 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     7201 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     7261 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     7321 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     7381 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     7441 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     7501 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     7561 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     7621 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     7681 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     7741 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     7801 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     7861 tcactata
//
