LOCUS       sequence-455288-        6799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETDuet-1: Rpn8(1-179)-PreScission-Strep, 
            H6-PreScission-Rpn11(2-239, Delta-78–81).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6799)
  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 6799)
  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: pAM59
FEATURES             Location/Qualifiers
     source          1..6799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    56..80
                     /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)."
     promoter        complement(81..99)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(127..150)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(151..174)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             718..740
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    755..779
                     /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)."
     promoter        complement(780..798)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(886..905)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1111..1188
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1189..2271
                     /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     complement(1208..1227)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2843..3034
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3028..3050)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3136..3275
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3191..3210
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3290..3307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3461..4049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3541..3560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4130..4222
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4223..4291,4292..5083)
                     /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     complement(4441..4460)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5198..5653)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5335..5356)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5547..5566
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5690..5737)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5733..5751
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5789..5833)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    5942..5966
                     /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)."
     promoter        complement(5967..5985)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(6756..6779)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(6792..6799,1..10))
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ggtgatggtg catatgtata tctccttctt atacttaact aatatactaa gatggggaat
       61 tgttatccgc tcacaattcc cctatagtga gtcgtattag agctcgaatt cttactcgat
      121 cccttatttt tcaaactgcg gatggctcca gggcccctgg aacagaactt ccagtacgtc
      181 tctcaataag tgttctacac caatttcttc agcttcttcg gcctcaatag tacaaggcaa
      241 atgaagaaac gtcttttctg tagacgtacc atcatcctta acttgctcaa tcgcgacata
      301 tgcatctgtt ggtaaaccaa caccttgttg tttgacatca acaattaata atagcggatt
      361 attctgagta tattttttaa acagctcatt aatcttgagg tcagaagccc ttaatttagg
      421 accactatga taccatccaa tgagtttttc cttggcatta atctttttac acatttcatt
      481 catgttttca atataattat ggtctaaaaa ccacacgtca gagtttttct catcttcttc
      541 aaacggtaag gcaaaggaat tagtgactct gatagtggaa ctgttagcat cacctaagat
      601 gacaccaacg catcttttgt tttcttttgt ctgcgtacgc tcataatgat ccaaagcaga
      661 tagcaaaact agtggtgcaa tggtaacttt ctcgtgttgt agagacccca tggtatatct
      721 ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc
      781 ctatagtgag tcgtattaat ttcgcgggat cgagatcgat ctcgatcctc tacgccggac
      841 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
      901 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
      961 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     1021 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     1081 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     1141 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     1201 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     1261 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     1321 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     1381 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     1441 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     1501 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     1561 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     1621 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     1681 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     1741 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     1801 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     1861 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     1921 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     1981 ggtgcggaca tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     2041 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     2101 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     2161 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2221 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2281 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     2341 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actagcatga tcgtgctcct
     2401 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     2461 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     2521 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     2581 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     2641 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     2701 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     2761 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     2821 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     2881 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     2941 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     3001 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3061 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3121 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     3181 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     3241 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     3301 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3361 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3421 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3481 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3541 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3601 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3661 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3721 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3781 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3841 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3901 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3961 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4021 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4081 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgacctat ttgtttattt
     4141 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4201 tcatgattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4261 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4321 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4381 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4441 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4501 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4561 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4621 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4681 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4741 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4801 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4861 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4921 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4981 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5041 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5101 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5161 gatccttttt gataatctca tgccatcgtg ccgccagaaa ttgtaaacgt taatattttg
     5221 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5281 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5341 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5401 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5461 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5521 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5581 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5641 ctacagggcg cgtcccattc gccaatccgg atatagttcc tcctttcagc aaaaaacccc
     5701 tcaagacccg tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag
     5761 cctaggttaa ttaagctgcg ctagtagacg agtccatgtg ctggcgttca aatttcgcag
     5821 cagcggtttc tttaccagac tcgagggtac cgacgtcagc gatcgcgtgg ccggccgata
     5881 tccaattgag atctgccata tgtatatctc cttcttatac ttaactaata tactaagatg
     5941 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gattatgcgg
     6001 ccgtgtacaa tacgattact ttctgttcga cttaagcatt atgcggccgc ttaagccaaa
     6061 tttgattctt ctttttcttc ataatcgtac atcttaagac ctgattgcca ctgttcttta
     6121 tgtaagttca ttaacatctt ggtttccttc gcggttttat gataatcgat atttaaagaa
     6181 tagtaatgcc tattcagacc gtgaattaag gcttgaatgt tggccttgtt caataagcct
     6241 gtgttggagg ttgtttgtct aggctctaag ttatttatca atgcgcccgt gtcaatcaat
     6301 ctaaaagcat cgatgacaac ttttccctta acggattgaa tagggtcaac aacgacagca
     6361 acagctctgc tgtttagttg ttcaaaagat ttttgagtat taacatcaac agaagatagc
     6421 caacagccaa accctggatg agagtggtac cagccaacga ccatttggtc tctgcccgtt
     6481 tgttttaaca tgtccatcat cttcgcttgg aaaacatcat cgacagctcc ggtacccgat
     6541 tgaggcatcg caaacacgtc cacaacgtta accgtataat catcgacaaa ctcacctaac
     6601 atcaacccca tgacttccat gggaacacca gctctaccat gctttagcat ctttaaaagc
     6661 gcaatcgaag aaatataaac agtttctttc gtatcgtcac ggccggtgtc cgcggacccc
     6721 accttactat tcatcatcaa tctctgtagt cgttcgggcc cctggaacag aacttccaga
     6781 gaaccgcttc catggtgat
//
