LOCUS       Exported                7469 bp ds-DNA     circular SYN 29-SEP-2022
DEFINITION  Plasmid for the expression and purification of GFP labelled p62. 
            Internal reference: SMC390.
ACCESSION   .
VERSION     .
KEYWORDS    pET-Duet1_6xHis-TEV-eGFP-P62
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7469)
  AUTHORS   Turco E, Savova A, Gere F, Ferrari L, Romanov J, Schuschnig M, 
            Martens S
  TITLE     Reconstitution defines the roles of p62, NBR1 and TAX1BP1 in 
            ubiquitin condensate formation and autophagy initiation.
  JOURNAL   Nat Commun. 2021 Sep 1;12(1):5212. doi: 10.1038/s41467-021-25572-w.
  PUBMED    34471133
REFERENCE   2  (bases 1 to 7469)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 29, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7469
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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..853
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             140..853
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(182..203)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(443..462)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     790..811
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        2266..2284
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2285..2309
                     /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             2418..2462
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2500..2518)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2514..2561
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2598..3053
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2685..2704)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2895..2916
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3168..4028)
                     /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     3791..3810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4029..4121)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4202..4790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4691..4710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4944..4961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4976..5115
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5041..5060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5201..5223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5217..5408)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5980..7062)
                     /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     7024..7043
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7063..7140)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7346..7365
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7453..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 cgaggagctg ttcaccgggg tggtgcccat
      181 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      241 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      301 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      361 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      421 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
      481 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
      541 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
      601 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
      661 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
      721 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
      781 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
      841 cgagctgtac aagtccggac tcagatctcg agctcaaatg gcgtcgctca ccgtgaaggc
      901 ctaccttctg ggcaaggagg acgcggcgcg cgagattcgc cgcttcagct tctgctgcag
      961 ccccgagcct gaggcggaag ccgaggctgc ggcgggtccg ggaccctgcg agcggctgct
     1021 gagccgggtg gccgccctgt tccccgcgct gcggcctggc ggcttccagg cgcactaccg
     1081 cgatgaggac ggggacttgg ttgccttttc cagtgacgag gaattgacaa tggccatgtc
     1141 ctacgtgaag gatgacatct tccgaatcta cattaaagag aaaaaagagt gccggcggga
     1201 ccaccgccca ccgtgtgctc aggaggcgcc ccgcaacatg gtgcacccca atgtgatctg
     1261 cgatggctgc aatgggcctg tggtaggaac ccgctacaag tgcagcgtct gcccagacta
     1321 cgacttgtgt agcgtctgcg agggaaaggg cttgcaccgg gggcacacca agctcgcatt
     1381 ccccagcccc ttcgggcacc tgtctgaggg cttctcgcac agccgctggc tccggaaggt
     1441 gaaacacgga cacttcgggt ggccaggatg ggaaatgggt ccaccaggaa actggagccc
     1501 acgtcctcct cgtgcagggg aggcccgccc tggccccacg gcagaatcag cttctggtcc
     1561 atcggaggat ccgagtgtga atttcctgaa gaacgttggg gagagtgtgg cagctgccct
     1621 tagccctctg ggcattgaag ttgatatcga tgtggagcac ggagggaaaa gaagccgcct
     1681 gacccccgtc tctccagaga gttccagcac agaggagaag agcagctcac agccaagcag
     1741 ctgctgctct gaccccagca agccgggtgg gaatgttgag ggcgccacgc agtctctggc
     1801 ggagcagatg aggaagatcg ccttggagtc cgaggggcgc cctgaggaac agatggagtc
     1861 ggataactgt tcaggaggag atgatgactg gacccatctg tcttcaaaag aagtggaccc
     1921 gtctacaggt gaactccagt ccctacagat gccagaatcc gaagggccaa gctctctgga
     1981 cccctcccag gagggaccca cagggctgaa ggaagctgcc ttgtacccac atctcccgcc
     2041 agaggctgac ccgcggctga ttgagtccct ctcccagatg ctgtccatgg gcttctctga
     2101 tgaaggcggc tggctcacca ggctcctgca gaccaagaac tatgacatcg gagcggctct
     2161 ggacaccatc cagtattcaa agcatccccc gccgttgtga gcggccgcat aatgcttaag
     2221 tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta
     2281 taggggaatt gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag
     2341 gagatataca tatggcagat ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg
     2401 gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact
     2461 cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat
     2521 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     2581 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2641 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2701 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2761 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2821 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2881 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2941 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3001 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttctggcgg
     3061 cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3121 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3181 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3241 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3301 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3361 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3421 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3481 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3541 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3601 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3661 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3721 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3781 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3841 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3901 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3961 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4021 atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt attgtctcat
     4081 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg accaaaatcc
     4141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4321 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4801 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4861 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4921 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4981 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5041 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5101 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5161 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5221 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5281 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5341 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5401 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5461 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5521 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5581 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5641 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5701 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5761 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5821 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc
     5881 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     5941 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6001 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6061 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     6121 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     6181 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     6241 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     6301 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     6361 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     6421 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     6481 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     6541 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     6601 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     6661 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     6721 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     6781 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     6841 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     6901 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6961 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     7021 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     7081 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     7141 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     7201 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     7261 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     7321 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     7381 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
     7441 atcccgcgaa attaatacga ctcactata
//
