LOCUS       sequence-430393-        7460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for the expression of mCherry labelled p62 (SMC Internal No. 
            391)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7460)
  AUTHORS   Wurzer B, Zaffagnini G, Fracchiolla D, Turco E, Abert C, Romanov J, 
            Martens S
  TITLE     Oligomerization of p62 allows for selection of ubiquitinated cargo 
            and isolation membrane during selective autophagy.
  JOURNAL   Elife. 2015 Sep 28;4:e08941. doi: 10.7554/eLife.08941.
  PUBMED    26413874
REFERENCE   2  (bases 1 to 7460)
  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: "Elife."; 
            date: "2015-09-28"; volume: "4:e08941. doi"; pages: " 
            10.7554/eLife.08941"
COMMENT     Sequence Label: pET-Duet1-His-TEV-mCherry-p62
FEATURES             Location/Qualifiers
     source          1..7460
                     /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"
     promoter        2257..2275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2276..2300
                     /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             2409..2453
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2491..2509)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2505..2552
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2589..3044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2676..2695)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2886..2907
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3159..3950,3951..4019))
                     /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     3782..3801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4020..4112)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4193..4781
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4682..4701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4935..4952
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4967..5106
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5032..5051)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5192..5214
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5208..5399)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5971..7053)
                     /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     7015..7034
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7054..7131)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7337..7356
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7444..7460,1..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 gagctcaaat ggcgtcgctc accgtgaagg cctaccttct
      901 gggcaaggag gacgcggcgc gcgagattcg ccgcttcagc ttctgctgca gccccgagcc
      961 tgaggcggaa gccgaggctg cggcgggtcc gggaccctgc gagcggctgc tgagccgggt
     1021 ggccgccctg ttccccgcgc tgcggcctgg cggcttccag gcgcactacc gcgatgagga
     1081 cggggacttg gttgcctttt ccagtgacga ggaattgaca atggccatgt cctacgtgaa
     1141 ggatgacatc ttccgaatct acattaaaga gaaaaaagag tgccggcggg accaccgccc
     1201 accgtgtgct caggaggcgc cccgcaacat ggtgcacccc aatgtgatct gcgatggctg
     1261 caatgggcct gtggtaggaa cccgctacaa gtgcagcgtc tgcccagact acgacttgtg
     1321 tagcgtctgc gagggaaagg gcttgcaccg ggggcacacc aagctcgcat tccccagccc
     1381 cttcgggcac ctgtctgagg gcttctcgca cagccgctgg ctccggaagg tgaaacacgg
     1441 acacttcggg tggccaggat gggaaatggg tccaccagga aactggagcc cacgtcctcc
     1501 tcgtgcaggg gaggcccgcc ctggccccac ggcagaatca gcttctggtc catcggagga
     1561 tccgagtgtg aatttcctga agaacgttgg ggagagtgtg gcagctgccc ttagccctct
     1621 gggcattgaa gttgatatcg atgtggagca cggagggaaa agaagccgcc tgacccccgt
     1681 ctctccagag agttccagca cagaggagaa gagcagctca cagccaagca gctgctgctc
     1741 tgaccccagc aagccgggtg ggaatgttga gggcgccacg cagtctctgg cggagcagat
     1801 gaggaagatc gccttggagt ccgaggggcg ccctgaggaa cagatggagt cggataactg
     1861 ttcaggagga gatgatgact ggacccatct gtcttcaaaa gaagtggacc cgtctacagg
     1921 tgaactccag tccctacaga tgccagaatc cgaagggcca agctctctgg acccctccca
     1981 ggagggaccc acagggctga aggaagctgc cttgtaccca catctcccgc cagaggctga
     2041 cccgcggctg attgagtccc tctcccagat gctgtccatg ggcttctctg atgaaggcgg
     2101 ctggctcacc aggctcctgc agaccaagaa ctatgacatc ggagcggctc tggacaccat
     2161 ccagtattca aagcatcccc cgccgttgtg agcggccgca taatgcttaa gtcgaacaga
     2221 aagtaatcgt attgtacacg gccgcataat cgaaattaat acgactcact ataggggaat
     2281 tgtgagcgga taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac
     2341 atatggcaga tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg
     2401 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     2461 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     2521 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2581 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2641 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2701 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2761 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2821 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2881 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2941 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3001 aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttctggcg gcacgatggc
     3061 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3181 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3241 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3301 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3361 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3421 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3481 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3541 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3601 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3661 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3721 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3781 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3841 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3901 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3961 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4021 ctcttccttt ttcaatcatg attgaagcat ttatcagggt tattgtctca tgagcggata
     4081 catatttgaa tgtatttaga aaaataaaca aataggtcat gaccaaaatc ccttaacgtg
     4141 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4201 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4261 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4321 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4381 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4441 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4501 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4561 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4621 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4681 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4741 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4801 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4861 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4921 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4981 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     5041 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     5101 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5161 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5221 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     5281 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5341 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     5401 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     5461 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     5521 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     5581 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     5641 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     5701 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     5761 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     5821 cctagccggg tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg
     5881 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5941 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6001 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6061 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     6121 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     6181 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     6241 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     6301 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     6361 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     6421 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6481 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     6541 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     6601 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     6661 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6721 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6781 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6841 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6901 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6961 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     7021 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     7081 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     7141 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     7201 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     7261 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     7321 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     7381 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
     7441 aattaatacg actcactata
//
