LOCUS       sequence-370664-        7650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the yeast Ulp1 Sumo protease in active form as a TEV 
            protease-cleavable MBP fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7650)
  AUTHORS   Mazzei L, Greene-Cramer R, Bafna K, Jovanovic A, De Falco A, Acton 
            TB, Royer CA, Ciurli S, Montelione GT
  TITLE     Protocol for production and purification of SARS-CoV-2 3CL(pro).
  JOURNAL   STAR Protoc. 2023 May 5;4(2):102326. doi: 
            10.1016/j.xpro.2023.102326.
  PUBMED    37235475
REFERENCE   2  (bases 1 to 7650)
  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: "STAR 
            Protoc."; date: "2023-05-5"; volume: "4(2):102326. doi"; pages: " 
            10.1016/j.xpro.2023.102326"
COMMENT     Sequence Label: pGTM_YR375_SUMO_Protease_001
FEATURES             Location/Qualifiers
     source          1..7650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      307..895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     796..815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1049..1066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1081..1220
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1146..1165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1306..1328
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1322..1513)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3868..3887
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3907..3984)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4190..4209
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4293..4312
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4293..4311
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4312..4336
                     /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             4351..4373
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4386..4409
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             4416..5513
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     5487..5510
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5538..5561
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5562..5582
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6471..6489)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6485..6532
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(6735..6753)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6821..6925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6926..6994,6995..7650,1..136)
                     /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(7144..7163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
       61 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
      121 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
      181 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
      241 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
      301 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
      361 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
      421 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
      481 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
      541 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
      601 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
      661 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
      721 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
      781 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
      841 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
      901 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
      961 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     1021 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     1081 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     1141 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     1201 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     1261 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     1321 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     1381 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     1441 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     1501 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     1561 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     1621 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     1681 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     1741 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     1801 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     1861 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     1921 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     1981 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     2041 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     2101 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     2161 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     2221 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     2281 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     2341 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     2401 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     2461 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     2521 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     2581 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     2641 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     2701 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     2761 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     2821 gcgctcactg cccgctttcc agtcgggaaa ctgtcgtgcc agctgcatta atgaatcggc
     2881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     2941 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     3001 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     3061 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     3121 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     3181 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     3241 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     3301 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     3361 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     3421 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     3481 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     3541 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     3601 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     3661 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     3721 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     3781 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     3841 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     3901 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     3961 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     4021 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     4081 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     4141 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     4201 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     4261 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     4321 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     4381 tgggacatca ccatcatcac catcaccatg catccaaaat cgaagaaggt aaactggtaa
     4441 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
     4501 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
     4561 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
     4621 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
     4681 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
     4741 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
     4801 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
     4861 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
     4921 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
     4981 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
     5041 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
     5101 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
     5161 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg
     5221 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
     5281 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     5341 aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
     5401 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     5461 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actttaatta
     5521 acggcgacgg tgccgggctg gaagttctgt tccaggggcc cgaaaacctg tattttcaga
     5581 gccatatgtc ctccctggaa cgcaaacaca aagactatct gaaccaaaaa ctgaaattcg
     5641 accgcagcat cctggaattc gaaaaagact tcaaacgtta caacgaaatc ctgaacgagc
     5701 gtaagaaaat tcaggaagat ctgaaaaaga aaaaagaaca actggcgaag aaaaaactgg
     5761 tgccggaact gaacgaaaaa gatgacgatc aggttcaaaa agcgctggcc agccgtgaaa
     5821 atacccagct gatgaaccgc gacaatattg aaatcaccgt gcgtgatttt aaaacgctgg
     5881 cgccgcgtcg ctggctgaac gataccatta tcgaattttt catgaaatac atcgaaaaat
     5941 ctaccccgaa cacggtcgcc ttcaatagct ttttctatac gaatctgtcc gaacgcggct
     6001 accagggtgt gcgtcgctgg atgaagcgta agaaaaccca aatcgacaaa ctggataaaa
     6061 tctttacgcc gatcaacctg aatcagtccc attgggcact gggcattatc gacctgaaaa
     6121 agaaaaccat tggctatgtt gatagcctgt ctaacggtcc gaatgcgatg tcatttgcca
     6181 tcctgaccga tctgcagaaa tacgtcatgg aagaatcgaa acatacgatt ggcgaagact
     6241 tcgatctgat ccacctggac tgcccgcagc aaccgaacgg ctatgattgc ggtatttacg
     6301 tttgtatgaa taccctgtat ggtagcgcag acgctccgct ggactttgat tacaaagatg
     6361 cgatccgcat gcgtcgcttc attgcacacc tgatcctgac ggatgctctg aaataactcg
     6421 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     6481 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     6541 aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     6601 cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata
     6661 cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta aagctaattc
     6721 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6781 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6841 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6901 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6961 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     7021 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     7081 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     7141 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     7201 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     7261 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     7321 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     7381 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     7441 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     7501 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     7561 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     7621 atctggagcc ggtgagcgtg ggtctcgcgg
//
