LOCUS       sequence-455296-        5513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28a Ubiquitin-G77C78.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5513)
  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 5513)
  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: pAM76
FEATURES             Location/Qualifiers
     source          1..5513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        79..97
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    98..122
                     /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             135..157
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             167..394
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             456..473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(526..544)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      540..587
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      624..1079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(711..730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     921..942
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1171..1986)
                     /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(1171..1986)
                     /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     1894..1913
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1978..1997
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2108..2696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2597..2616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2850..2867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2882..3021
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2947..2966)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3107..3129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3123..3314)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4123..5205)
                     /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     5167..5186
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5206..5283)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5489..5508
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
       61 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
      121 cccctctgaa taattttgtt taactttaag aaggagatat acccatatgc agattttcgt
      181 caagactttg accggtaaaa ccataacatt ggaagttgaa tcttccgata ccatcgacaa
      241 cgttaagtcg aaaattcaag acaaggaagg tatccctcca gatcaacaaa gattgatctt
      301 tgccggtaag cagctagaag acggtagaac gctgtctgat tacaacattc agaaggagtc
      361 caccttacat cttgtgctaa ggctaagagg tggtggctgc taatgatgag gatccgaatt
      421 cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
      481 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      541 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      601 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      661 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      721 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      781 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
      841 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
      901 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
      961 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1021 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1081 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1141 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     1201 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     1261 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     1321 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     1381 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     1441 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     1501 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     1561 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     1621 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     1681 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     1741 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     1801 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     1861 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     1921 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     1981 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     2041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2221 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2701 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2761 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2821 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2881 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     2941 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3001 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3061 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3121 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3181 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3241 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3301 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3361 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3421 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3481 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3541 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3601 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3661 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3721 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     3781 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     3841 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     3901 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     3961 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4021 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4081 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4141 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4201 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4261 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4321 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4381 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4441 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4501 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4561 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4621 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4681 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     4741 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4801 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4861 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     4921 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     4981 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5041 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5101 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5161 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5221 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5281 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5341 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5401 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5461 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg cca
//
