LOCUS       sequence-451487-        5522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28a MWG-ubiquitin-G77-C78.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5522)
  AUTHORS   Xie G, Dong KC, Worden EJ, Martin A
  TITLE     High-Throughput Assay for Characterizing Rpn11 Deubiquitinase 
            Activity.
  JOURNAL   Methods Mol Biol. 2023;2591:79-100. doi: 
            10.1007/978-1-0716-2803-4_6.
  PUBMED    36350544
REFERENCE   2  (bases 1 to 5522)
  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: "Methods Mol
            Biol."; date: "2023"; volume: "2591:79-100. doi"; pages: " 
            10.1007/978-1-0716-2803-4_6"
COMMENT     Sequence Label: pAM75
FEATURES             Location/Qualifiers
     source          1..5522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             189..206
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(259..277)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      273..320
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      357..812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(444..463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     654..675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(904..1719)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(904..1719)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1627..1646
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1711..1730
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1841..2429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2330..2349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2583..2600
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2615..2754
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2680..2699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2840..2862
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2856..3047)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3856..4938)
                     /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     4900..4919
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4939..5016)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5222..5241
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5325..5344
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5325..5343
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5344..5368
                     /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             5381..5403
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(5422..5522,1..127)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
ORIGIN
        1 aggtatccct ccagatcaac aaagattgat ctttgccggt aagcagctag aagacggtag
       61 aacgctgtct gattacaaca ttcagaagga gtccacctta catcttgtgc taaggctaag
      121 aggtggtggc tgctaatgat gaggatccga attcgagctc cgtcgacaag cttgcggccg
      181 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
      241 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      301 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
      361 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      421 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      481 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      541 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
      601 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
      661 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
      721 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
      781 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
      841 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
      901 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
      961 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1021 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     1081 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     1141 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     1201 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     1261 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     1321 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     1381 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     1441 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     1501 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     1561 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     1621 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     1681 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     1741 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1801 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1861 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1921 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1981 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2041 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2101 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2161 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2221 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2281 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2341 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2401 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2461 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2521 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2581 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     2641 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     2701 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2761 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2821 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2881 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2941 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3001 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3061 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3121 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3181 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3241 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3301 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3361 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3421 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     3481 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     3541 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     3601 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     3661 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     3721 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     3781 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     3841 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3901 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3961 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4021 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4081 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4141 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4201 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4261 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4321 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4381 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4441 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4501 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4561 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4621 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4681 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4741 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4801 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4861 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4921 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4981 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5041 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5101 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5161 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5221 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5281 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     5341 aggggaattg tgagcggata acaattcccc tctgaataat tttgtttaac tttaagaagg
     5401 agatataccc atatgtgggg catgcagatt ttcgtcaaga ctttgaccgg taaaaccata
     5461 acattggaag ttgaatcttc cgataccatc gacaacgtta agtcgaaaat tcaagacaag
     5521 ga
//
