LOCUS       sequence-446622-        6391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Ufd1L55R-3C-His6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6391)
  AUTHORS   Williams C, Dong KC, Arkinson C, Martin A
  TITLE     The Ufd1 cofactor determines the linkage specificity of 
            polyubiquitin chain engagement by the AAA+ ATPase Cdc48.
  JOURNAL   Mol Cell. 2023 Mar 2;83(5):759-769.e7. doi: 
            10.1016/j.molcel.2023.01.016. Epub 2023 Feb 2.
  PUBMED    36736315
REFERENCE   2  (bases 1 to 6391)
  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.2023.01.016"; journalName: "Mol Cell"; date: 
            "2023-03-2- 2"; volume: "83"; issue: "5"; pages: "759-769.e7"
COMMENT     Sequence Label: pAM296
FEATURES             Location/Qualifiers
     source          1..6391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(514..546)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             554..576
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    591..615
                     /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)."
     primer_bind     complement(615..634)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(616..634)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(718..737)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        943..1020
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1021..2103
                     /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     complement(1040..1059)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2912..3103
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3097..3119)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3205..3344
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3260..3279
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3359..3376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3530..4118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3610..3629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4229..4248)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4240..5055
                     /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             4240..5055
                     /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     complement(4313..4332)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5147..5602)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5284..5305)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5496..5515
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5639..5686)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5682..5700
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5753..5770)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5777..5800)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 gtaactaatc tcaataacat cgtccacggt caaagtcgag aaattcctca gtacgttttc
       61 caaaactgct ttggggtcgg aaatatccaa gaaatctaca gactgaggtt ctaacttcac
      121 aaactggccc agaggtacat cagtagatga aatttgcagt agagatcctg gctgtatacc
      181 caaagtttcc atcatccatt gtggtaagta aactctgccc tcttcagcaa taaattctaa
      241 aacacctcca tgtgtcacac gtccagtttc attggcagtt agtttaaaaa gcatggggta
      301 tcttatgttc aacatggata gtttgctacg agcacttggt ggcaggaata ttttcccgcc
      361 gaagtttgcg tcatccttcc gtattctatc attcatcata gctatagggt agcatctaaa
      421 gaactcctca aacgtttgag gcatatttac aaaaccattt ccgccgccaa aagaactaaa
      481 gccagaaaac atgagctcga attcggatcc gcgacccatt tgctgtccac cagtcatgct
      541 agccatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
      601 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
      661 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
      721 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
      781 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
      841 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
      901 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
      961 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     1021 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     1081 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     1141 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     1201 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     1261 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     1321 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     1381 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     1441 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     1501 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     1561 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     1621 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     1681 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     1741 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     1801 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     1861 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     1921 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     1981 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     2041 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2101 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     2161 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     2221 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     2281 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     2341 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     2401 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     2461 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     2521 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     2581 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     2641 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     2701 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     2761 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     2821 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     2881 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     2941 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     3001 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     3061 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3121 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3181 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3241 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     3301 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     3361 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3421 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3481 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3541 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3601 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3661 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3721 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3781 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3841 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3901 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3961 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4021 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4081 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4141 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     4201 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     4261 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     4321 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     4381 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     4441 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     4501 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     4561 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     4621 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     4681 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     4741 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     4801 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     4861 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     4921 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     4981 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     5041 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     5101 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5161 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5221 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5281 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5341 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5401 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5461 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5521 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5581 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     5641 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     5701 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt
     5761 ggtggtggtg ctcgaggggc ccctggaaca gaacttccag gtcaatttcg attacttccg
     5821 ggcttttggg agccttggac ttggacgaat catggtcact tttcgttttt ctcttattgc
     5881 tcttccttag tgagatccct tggccttgga aattttgttc actggatttg ctaccagcgg
     5941 cactttcttc gtcttctttc ggtaggacca ttgggaaacc aaagaataat tggccttcag
     6001 gcagatctag cttggcgggc tcgccatcaa acgttatctt catatctttt atatcttgct
     6061 tgggctccgc tttcggagct ttaccagata tgttttgtcc ctgacctaca aatttagata
     6121 gtttgtttct gctactgtta gcgatacccg catagtcgat tcttgtggac atcgatccct
     6181 ggccaagcac cgagggatcc aaaacttggc ccttaccaaa agaattcttt ttttctttat
     6241 cttgctgagc tttcagcgct ttgtaatccg gttccacata tcctacaggt ggagcaaaat
     6301 ctgttactaa atctgtttca atgacacata tactcttcga tgatgattct ggtttgactt
     6361 ctaagatctt aattttgaaa gttttaccat t
//
