LOCUS       sequence-252682-        6708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-RPN13 (19-132) K97E in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6708)
  AUTHORS   Hemmis CW, Heard SC, Hill CP
  TITLE     Phosphorylation of Tyr950 in the proteasome scaffolding protein RPN2
            modulates its interaction with the ubiquitin receptor RPN13.
  JOURNAL   J Biol Chem. 2019 May 7. pii: AC119.008881. doi: 
            10.1074/jbc.AC119.008881.
  PUBMED    31064842
REFERENCE   2  (bases 1 to 6708)
  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: "J Biol 
            Chem. 2019 May 7. pii: AC119.008881. doi: 10.1074/jbc.AC119.008881."
COMMENT     Sequence Label: pET151-GST-PP-RPN13 (19-132) K97E
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             91..744
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             751..774
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1182..1200)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1196..1243
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1481..1499)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1567..1671
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1672..1740,1741..2532)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1890..1909)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2703..3291
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3192..3211
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3445..3462
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3477..3616
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3542..3561)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3702..3724
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3718..3909)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5221..6303)
                     /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     6265..6284
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6304..6381)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6587..6606
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6690..6708,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6690..6708
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgggaattga tcccttcacc atgtccccta tactaggtta ttggaaaatt
      121 aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa atatgaagag
      181 catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga attgggtttg
      241 gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca gtctatggcc
      301 atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa agagcgtgca
      361 gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc gagaattgca
      421 tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc tgaaatgctg
      481 aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca tgtaacccat
      541 cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc aatgtgcctg
      601 gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc acaaattgat
      661 aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca agccacgttt
      721 ggtggtggcg accatcctcc aaaatcggat ctggaagttc tgttccaggg gccgtccaac
      781 aagtacttgg tggagtttcg ggcgggaaag atgtccctga aggggaccac cgtgactccg
      841 gataagcgga aagggctggt gtacattcag cagacggacg actcgcttat tcacttctgc
      901 tggaaggaca ggacgtccgg gaacgtggaa gacgacttga tcatcttccc tgacgactgt
      961 gagttcaagc gggtgccgca gtgccccagc gggagggtct acgtgctgga gttcaaggca
     1021 gggtccaagc ggcttttctt ctggatgcag gaacccaaga cagaccagga tgaggagcat
     1081 tgccggaaag tcaacgagta tctgaacaac cccccgtaaa agggcgagct cagatccggc
     1141 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1201 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1261 atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca
     1321 tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc
     1381 tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagctagct tatcgatgat
     1441 aagctgtcaa acatgagaat taattcttga agacgaaagg gcctcgtgat acgcctattt
     1501 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     1561 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1621 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1681 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1741 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1801 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1861 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     1921 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     1981 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2041 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2101 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2161 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     2221 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2281 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2341 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2401 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2461 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2521 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2581 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2641 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2701 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2761 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2821 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2881 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2941 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3001 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3061 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3121 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3181 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3241 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3301 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3361 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3421 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3481 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     3541 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3601 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3661 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3721 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3781 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     3841 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     3901 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     3961 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4021 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4081 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4141 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4201 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4261 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4321 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     4381 ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga
     4441 tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc
     4501 aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg
     4561 gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
     4621 acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc
     4681 agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc
     4741 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     4801 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc
     4861 aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4921 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4981 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5041 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5101 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5161 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5221 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5281 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5341 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5401 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     5461 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     5521 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     5581 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5641 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5701 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     5761 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5821 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5881 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5941 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6001 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6061 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6121 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6181 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6241 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6301 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6361 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6421 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     6481 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6541 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6601 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6661 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagg
//
