LOCUS       sequence-455206-        6465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETDuet-1 H6-precission-Rpn11(24-160-GGSGSG-192-221) , Rpn8(1-179).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6465)
  AUTHORS   Worden EJ, Dong KC, Martin A
  TITLE     An AAA Motor-Driven Mechanical Switch in Rpn11 Controls 
            Deubiquitination at the 26S Proteasome.
  JOURNAL   Mol Cell. 2017 Sep 7;67(5):799-811.e8. doi: 
            10.1016/j.molcel.2017.07.023. Epub 2017 Aug 24.
  PUBMED    28844860
REFERENCE   2  (bases 1 to 6465)
  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: 
            "10.1016/j.molcel.2017.07.023"; journalName: "Mol Cell"; date: 
            "2017-09-7- 7"; volume: "67"; issue: "5"; pages: "799-811.e8"
COMMENT     Sequence Label: pAM71
FEATURES             Location/Qualifiers
     source          1..6465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     597..616
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(636..713)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     919..938
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1026..1044
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1045..1069
                     /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             1084..1106
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1125..1142
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1155..1178
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1806..1824
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1825..1849
                     /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             2456..2500
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2538..2556)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2552..2599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2636..3091
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2723..2742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2933..2954
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3206..3997,3998..4066))
                     /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     3829..3848
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4067..4159)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4240..4828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4729..4748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4982..4999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5014..5153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5079..5098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5239..5261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5255..5446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6018..6465,1..635))
                     /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 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
       61 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
      121 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
      181 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
      241 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
      301 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
      361 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
      421 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
      481 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
      541 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
      601 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
      661 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
      721 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
      781 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
      841 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
      901 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
      961 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc
     1021 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     1081 aattttgttt aactttaaga aggagatata ccatgggcag cagccatcac catcatcacc
     1141 acagccagga tccgctggaa gttctgttcc aggggcccat gaaagaaact gtttatattt
     1201 cttcgattgc gcttttaaag atgctaaagc atggtagagc tggtgttccc atggaagtca
     1261 tggggttgat gttaggtgag tttgtcgatg attatacggt taacgttgtg gacgtgtttg
     1321 cgatgcctca atcgggtacc ggagtttctg ttgaggctgt cgatgatgtt ttccaagcga
     1381 agatgatgga catgttaaaa caaacgggca gagaccaaat ggtcgttggc tggtaccact
     1441 ctcatccagg gtttggctgt tggctatctt ctgttgatgt taatactcaa aaatcttttg
     1501 aacaactaaa cagcagagct gttgctgtcg ttgttgaccc tattcaatcc gttaagggaa
     1561 aagttgtcat cgatgctttt agattgattg acggcggttc tggttctggt ctgaataggc
     1621 attactattc tttaaatatc gattatcata aaaccgcgaa ggaaaccaag atgttaatga
     1681 acttacataa agaacagtgg caatcataag agctcggcgc gcctgcaggt cgacaagctt
     1741 gcggccgcat aatgcttaag tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc
     1801 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc catcttagta
     1861 tattagttaa gtataagaag gagatataca tatgatgtct ctacaacacg agaaagttac
     1921 cattgcacca ctagttttgc tatctgcttt ggatcattat gagcgtacgc agacaaaaga
     1981 aaacaaaaga tgcgttggtg tcatcttagg tgatgctaac agttccacta tcagagtcac
     2041 taattccttt gccttaccgt ttgaagaaga tgagaaaaac tctgacgtgt ggtttttaga
     2101 ccataattat attgaaaaca tgaatgaaat gtgtaaaaag attaatgcca aggaaaaact
     2161 cattggatgg tatcatagtg gtcctaaatt aagggcttct gacctcaaga ttaatgagct
     2221 gtttaaaaaa tatactcaga ataatccgct attattaatt gttgatgtca aacaacaagg
     2281 tgttggttta ccaacagatg catatgtcgc gattgagcaa gttaaggatg atggtacgtc
     2341 tacagaaaag acgtttcttc atttgccttg tactattgag gccgaagaag ctgaagaaat
     2401 tggtgtagaa cacttattga gagacgtata agacgtcggt accctcgagt ctggtaaaga
     2461 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     2521 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2581 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     2641 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2701 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2761 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2821 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2881 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2941 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3001 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3061 aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg agattatcaa
     3121 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3181 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3241 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3301 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3361 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3421 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3481 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3541 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3601 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3661 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3721 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3781 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3841 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3901 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3961 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4021 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4081 aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4141 atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt tttcgttcca
     4201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5041 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     5101 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     5161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5281 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     5341 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5401 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5461 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     5521 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5581 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     5641 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     5701 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     5761 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     5821 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     5881 tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag gagctgactg
     5941 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     6001 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6061 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     6121 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6181 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6241 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     6301 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6361 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6421 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatc
//
