LOCUS       sequence-373590-        6749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the protein expression of 6xHis-TEV-NDP52 in a bacterial
            expression system. Internal reference: SMC401.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6749)
  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 6749)
  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-Duet1-6xHis-TEV-NDP52
FEATURES             Location/Qualifiers
     source          1..6749
                     /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"
     promoter        1546..1564
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1565..1589
                     /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..1742
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1780..1798)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1794..1841
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1878..2333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1965..1984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2175..2196
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2448..3239,3240..3308))
                     /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     3071..3090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3309..3401)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3482..4070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3971..3990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4224..4241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4256..4395
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4321..4340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4481..4503
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4497..4688)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5260..6342)
                     /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     6304..6323
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6343..6420)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6626..6645
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6733..6749,1..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 tttcagggaa tggaggagac catcaaagat ccccccacat cagctgtctt
      181 gctggatcac tgtcatttct ctcaggtcat ctttaacagt gtggagaagt tctacatccc
      241 tggaggggac gtcacatgtc attatacctt cacccagcat ttcatccctc gtcgaaagga
      301 ttggattggc atctttagag tggggtggaa gacaacccgt gagtattaca ccttcatgtg
      361 ggttactttg cccattgacc taaacaacaa atcagctaaa cagcaggaag tccaattcaa
      421 agcttactac ctgcccaagg atgatgagta ttaccagttc tgctatgtgg atgaggatgg
      481 tgtggtccgg ggagcaagta ttcctttcca attccgtcca gaaaatgagg aagacatcct
      541 ggttgttacc actcagggag aggtggaaga gattgagcag cacaacaagg agctttgcaa
      601 agaaaaccag gagctgaagg acagctgtat cagcctccag aagcagaact cagacatgca
      661 ggctgagctc caaaagaagc aggaggagct agaaacccta cagagcatca ataagaagtt
      721 ggaactgaaa gtgaaagaac agaaggacta ttgggagaca gagctgcttc aactgaaaga
      781 acaaaaccag aagatgtcct cagaaaatga gaagatggga atcagagtgg atcagcttca
      841 ggcccagctg tcaactcaag agaaagaaat ggagaagctt gttcagggag atcaagataa
      901 gacagagcag ttagagcagc tgaaaaagga aaatgaccac ctctttctca gtttaactga
      961 acagaggaag gaccagaaga agctcgagca gacagtggag caaatgaagc agaatgaaac
     1021 tactgcaatg aagaaacaac aggaattaat ggatgaaaac tttgacctgt caaaaagact
     1081 gagtgagaac gaaattatat gtaatgctct gcagagacag aaagagagat tggaaggaga
     1141 aaatgatctt ttgaagaggg agaacagcag attgctcagt tacatgggtc tggattttaa
     1201 ttctttgccg tatcaagtac ctacttcaga tgaaggaggc gcaagacaaa atccaggact
     1261 tgcctatgga aacccatatt ctggtatcca agaaagttct tccgccagcc cgctctccat
     1321 caagaaatgc cctatctgca aagcagatga tatttgtgat cacaccttgg agcaacagca
     1381 gatgcagccc ctttgtttca attgtccaat ttgtgacaag atcttcccag ctacagagaa
     1441 gcagatcttt gaagaccacg tgttctgcca ctctctctga gcggccgcat aatgcttaag
     1501 tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta
     1561 taggggaatt gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag
     1621 gagatataca tatggcagat ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg
     1681 gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact
     1741 cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat
     1801 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1861 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1921 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1981 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2041 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2101 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2161 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2221 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2281 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttctggcgg
     2341 cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2401 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2461 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2521 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2581 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2641 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2701 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2761 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2821 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2881 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2941 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3001 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3061 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3121 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3181 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3241 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3301 atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt attgtctcat
     3361 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg accaaaatcc
     3421 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3481 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3541 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3601 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3661 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3721 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3781 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3841 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3901 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3961 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4021 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4081 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4141 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4201 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4261 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4321 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4381 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4441 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4501 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4561 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4621 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4681 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4741 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4801 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4861 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4921 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4981 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5041 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5101 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc
     5161 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     5221 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5281 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5341 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     5401 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     5461 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     5521 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     5581 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     5641 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5701 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5761 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5821 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5881 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5941 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     6001 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     6061 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     6121 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     6181 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6241 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     6301 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     6361 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     6421 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     6481 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     6541 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     6601 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     6661 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
     6721 atcccgcgaa attaatacga ctcactata
//
