LOCUS       sequence-245573-        6708 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses GST-RPN13 (19-132) R104E 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 Apr 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) R104E
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             110..763
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             770..793
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1201..1219)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1215..1262
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1500..1518)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1586..1690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1691..1759,1760..2551)
                     /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(1909..1928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2722..3310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3211..3230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3464..3481
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3496..3635
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3561..3580)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3721..3743
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3737..3928)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5240..6322)
                     /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     6284..6303
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6323..6400)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6606..6625
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggaattgat cccttcacca tgtcccctat
      121 actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct
      181 tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa
      241 aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa
      301 attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg
      361 ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata
      421 cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag
      481 caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa
      541 tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata
      601 catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga
      661 agctatccca caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca
      721 gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggaagttct
      781 gttccagggg ccgtccaaca agtacttggt ggagtttcgg gcgggaaaga tgtccctgaa
      841 ggggaccacc gtgactccgg ataagcggaa agggctggtg tacattcagc agacggacga
      901 ctcgcttatt cacttctgct ggaaggacag gacgtccggg aacgtggaag acgacttgat
      961 catcttccct gacgactgtg agttcaagcg ggtgccgcag tgccccagcg ggagggtcta
     1021 cgtgctgaag ttcaaggcag ggtccaagga gcttttcttc tggatgcagg aacccaagac
     1081 agaccaggat gaggagcatt gccggaaagt caacgagtat ctgaacaacc ccccgtaaaa
     1141 gggcgagctc agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1201 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1261 tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac
     1321 caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt
     1381 agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta
     1441 aagctagctt atcgatgata agctgtcaaa catgagaatt aattcttgaa gacgaaaggg
     1501 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     1561 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1621 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1681 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1741 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1801 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1861 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1921 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1981 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2041 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2101 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2161 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2221 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2281 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2341 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2401 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2461 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2521 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2581 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2641 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2701 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2761 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2821 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2881 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2941 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3001 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3061 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3121 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3181 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3241 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3301 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3361 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3421 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3481 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3541 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3601 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3661 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3721 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3781 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3841 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3901 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3961 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4021 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4081 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4141 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4201 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4261 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4321 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4381 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     4441 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     4501 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     4561 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     4621 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     4681 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     4741 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     4801 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     4861 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     4921 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4981 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5041 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5101 gaagacagtc ataagtgcgg cgacaatagt catgccccgc gcccaccgga aggagctgac
     5161 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5221 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5281 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5341 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5401 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5461 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5521 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5581 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5641 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5701 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5761 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5821 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5881 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5941 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6001 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6061 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6121 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6181 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6241 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6301 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6361 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6421 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6481 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6541 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6601 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6661 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
