LOCUS       sequence-373595-        7142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression of 6xHis-TEV-OPTN. Internal reference: 
            SMC397.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7142)
  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 7142)
  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-OPTN
FEATURES             Location/Qualifiers
     source          1..7142
                     /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             457..468
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        1939..1957
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1958..1982
                     /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             2091..2135
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2173..2191)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2187..2234
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2271..2726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2358..2377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2568..2589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2841..3632,3633..3701))
                     /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     3464..3483
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3702..3794)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3875..4463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4364..4383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4617..4634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4649..4788
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4714..4733)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4874..4896
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4890..5081)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5653..6735)
                     /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     6697..6716
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6736..6813)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7019..7038
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7126..7142,1..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 tgtcccatca acctctcagc tgcctcactg aaaaggagga
      181 cagccccagt gaaagcacag gaaatggacc cccccacctg gcccacccaa acctggacac
      241 gtttaccccg gaggagctgc tgcagcagat gaaagagctc ctgaccgaga accaccagct
      301 gaaagaagcc atgaagctaa ataatcaagc catgaaaggg agatttgagg agctttcggc
      361 ctggacagag aaacagaagg aagaacgcca gttttttgag atacagagca aagaagcaaa
      421 agagcgtcta atggccttga gtcatgagaa tgagaaattg aaggaagagc ttggaaaact
      481 aaaagggaaa tcagaaaggt catctgagga ccccactgat gactccaggc ttcccagggc
      541 cgaagcggag caggaaaagg accagctcag gacccaggtg gtgaggctac aagcagagaa
      601 ggcagacctg ttgggcatcg tgtctgaact gcagctcaag ctgaactcca gcggctcctc
      661 agaagattcc tttgttgaaa ttaggatggc tgaaggagaa gcagaagggt cagtaaaaga
      721 aatcaagcat agtcctgggc ccacgagaac agtctccact ggcacggcat tgtctaaata
      781 taggagcaga tctgcagatg gggccaagaa ttacttcgaa catgaggagt taactgtgag
      841 ccagctcctg ctgtgcctaa gggaagggaa tcagaaggtg gagagacttg aagttgcact
      901 caaggaggcc aaagaaagag tttcagattt tgaaaagaaa acaagtaatc gttctgagat
      961 tgaaacccag acagagggga gcacagagaa agagaatgat gaagagaaag gcccggagac
     1021 tgttggaagc gaagtggaag cactgaacct ccaggtgaca tctctgttta aggagcttca
     1081 agaggctcat acaaaactca gcgaagctga gctaatgaag aagagacttc aagaaaagtg
     1141 tcaggccctt gaaaggaaaa attctgcaat tccatcagag ttgaatgaaa agcaagagct
     1201 tgtttatact aacaaaaagt tagagctaca agtggaaagc atgctatcag aaatcaaaat
     1261 ggaacaggct aaaacagagg atgaaaagtc caaattaact gtgctacaga tgacacacaa
     1321 caagcttctt caagaacata ataatgcatt gaaaacaatt gaggaactaa caagaaaaga
     1381 gtcagaaaaa gtggacaggg cagtgctgaa ggaactgagt gaaaaactgg aactggcaga
     1441 gaaggctctg gcttccaaac agctgcaaat ggatgaaatg aagcaaacca ttgccaagca
     1501 ggaagaggac ctggaaacca tgaccatcct cagggctcag atggaagttt actgttctga
     1561 ttttcatgct gaaagagcag cgagagagaa aattcatgag gaaaaggagc aactggcatt
     1621 gcagctggca gttctgctga aagagaatga tgctttcgaa gacggaggca ggcagtcctt
     1681 gatggagatg cagagtcgtc atggggcgag aacaagtgac tctgaccagc aggcttacct
     1741 tgttcaaaga ggagctgagg acagggactg gcggcaacag cggaatattc cgattcattc
     1801 ctgccccaag tgtggagagg ttctgcctga catagacacg ttacagattc acgtgatgga
     1861 ttgcatcatt taagcggccg cataatgctt aagtcgaaca gaaagtaatc gtattgtaca
     1921 cggccgcata atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1981 ccccatctta gtatattagt taagtataag aaggagatat acatatggca gatctcaatt
     2041 ggatatcggc cggccacgcg atcgctgacg tcggtaccct cgagtctggt aaagaaaccg
     2101 ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct
     2161 aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2221 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2281 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2341 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2401 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2461 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2521 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2581 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2641 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2701 taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt atcaaaaagg
     2761 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2821 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2881 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2941 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3001 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3061 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3121 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3181 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3241 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3301 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3361 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3421 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3481 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3541 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3601 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3661 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatca
     3721 tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3781 gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3841 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3901 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3961 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4021 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4081 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4141 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4201 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4261 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4321 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4381 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4441 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4501 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4561 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4621 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4681 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4741 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4801 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4861 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4921 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4981 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5041 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5101 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5161 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5221 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5281 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5341 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5401 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5461 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5521 gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5581 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5641 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5701 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5761 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5821 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5881 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     5941 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6001 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6061 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6121 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6181 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6241 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6301 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6361 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6421 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6481 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6541 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6601 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6661 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6721 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6781 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6841 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6901 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6961 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     7021 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     7081 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
     7141 ta
//
