LOCUS       sequence-455251-        6811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETDuet-1: Rpn8(1-179)-PreScission-Strep, 
            H6-PreScission-Rpn11(2-239, L132A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6811)
  AUTHORS   Worden EJ, Padovani C, Martin A
  TITLE     Structure of the Rpn11-Rpn8 dimer reveals mechanisms of substrate 
            deubiquitination during proteasomal degradation.
  JOURNAL   Nat Struct Mol Biol. 2014 Mar;21(3):220-7. doi: 10.1038/nsmb.2771. 
            Epub 2014 Jan 23.
  PUBMED    24463465
REFERENCE   2  (bases 1 to 6811)
  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"; doi: "10.1038/nsmb.2771";
            journalName: "Nat Struct Mol Biol"; date: "2014-03"; volume: "21"; 
            issue: "3"; pages: "220-7"
COMMENT     Sequence Label: pAM54
FEATURES             Location/Qualifiers
     source          1..6811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     424..443
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(463..540)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     746..765
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        853..871
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    872..896
                     /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             911..933
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1477..1500
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1501..1524
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        1552..1570
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1571..1595
                     /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             1641..1658
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1671..1694
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        2477..2495
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2496..2520
                     /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             2629..2673
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2711..2729)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2725..2772
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2809..3264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2896..2915)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3106..3127
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3379..4170,4171..4239))
                     /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     4002..4021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4240..4332)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4413..5001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4902..4921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5155..5172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5187..5326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5252..5271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5412..5434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5428..5619)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6191..6811,1..462))
                     /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"
ORIGIN
        1 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
       61 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      121 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      181 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      241 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      301 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
      361 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
      421 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
      481 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
      541 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
      601 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      661 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      721 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      781 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
      841 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      901 tagaaataat tttgtttaac tttaagaagg agatatacca tggggtctct acaacacgag
      961 aaagttacca ttgcaccact agttttgcta tctgctttgg atcattatga gcgtacgcag
     1021 acaaaagaaa acaaaagatg cgttggtgtc atcttaggtg atgctaacag ttccactatc
     1081 agagtcacta attcctttgc cttaccgttt gaagaagatg agaaaaactc tgacgtgtgg
     1141 tttttagacc ataattatat tgaaaacatg aatgaaatgt gtaaaaagat taatgccaag
     1201 gaaaaactca ttggatggta tcatagtggt cctaaattaa gggcttctga cctcaagatt
     1261 aatgagctgt ttaaaaaata tactcagaat aatccgctat tattaattgt tgatgtcaaa
     1321 caacaaggtg ttggtttacc aacagatgca tatgtcgcga ttgagcaagt taaggatgat
     1381 ggtacgtcta cagaaaagac gtttcttcat ttgccttgta ctattgaggc cgaagaagct
     1441 gaagaaattg gtgtagaaca cttattgaga gacgtactgg aagttctgtt ccaggggccc
     1501 tggagccatc cgcagtttga aaaataaggg atcgagtaag aattcgagct ctaatacgac
     1561 tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat
     1621 aagaaggaga tatacatatg caccatcacc atcaccatgg aagcggttct ctggaagttc
     1681 tgttccaggg gcccgaacga ctacagagat tgatgatgaa tagtaaggtg gggtccgcgg
     1741 acaccggccg tgacgatacg aaagaaactg tttatatttc ttcgattgcg cttttaaaga
     1801 tgctaaagca tggtagagct ggtgttccca tggaagtcat ggggttgatg ttaggtgagt
     1861 ttgtcgatga ttatacggtt aacgttgtgg acgtgtttgc gatgcctcaa tcgggtaccg
     1921 gagtttctgt tgaggctgtc gatgatgttt tccaagcgaa gatgatggac atgttaaaac
     1981 aaacgggcag agaccaaatg gtcgttggct ggtaccactc tcatccaggg tttggctgtt
     2041 ggctatcttc tgttgatgtt aatactcaaa aatcttttga acaagcgaac agcagagctg
     2101 ttgctgtcgt tgttgaccct attcaatccg ttaagggaaa agttgtcatc gatgctttta
     2161 gattgattga cacgggcgca ttgataaata acttagagcc tagacaaaca acctccaaca
     2221 caggcttatt gaacaaggcc aacattcaag ccttaattca cggtctgaat aggcattact
     2281 attctttaaa tatcgattat cataaaaccg cgaaggaaac caagatgtta atgaacttac
     2341 ataaagaaca gtggcaatca ggtcttaaga tgtacgatta tgaagaaaaa gaagaatcaa
     2401 atttggctta agcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg
     2461 gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     2521 ccatcttagt atattagtta agtataagaa ggagatatac atatggcaga tctcaattgg
     2581 atatcggccg gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa agaaaccgct
     2641 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     2701 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2761 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2821 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2881 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2941 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3001 acctcgaccc caaaaaactt gattagggtg atggttcaca tagtgggcca tcgccctgat
     3061 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3121 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3181 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3241 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     3301 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3361 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3421 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3481 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3541 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3601 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3661 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3721 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3781 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3841 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3901 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3961 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4021 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4081 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4141 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4201 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     4261 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4321 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4381 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4441 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4501 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4561 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4621 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4681 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4741 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4801 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4861 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4921 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4981 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5041 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5101 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5161 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5221 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5281 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5341 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5401 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5461 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5521 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5581 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5641 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5701 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5761 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5821 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5881 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5941 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     6001 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     6061 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     6121 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     6181 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6241 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     6301 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     6361 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     6421 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     6481 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     6541 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     6601 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     6661 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     6721 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     6781 cttcatggga gaaaataata ctgttgatgg g
//
