LOCUS       sequence-446598-        6391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Ufd1R64C-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: pAM295
FEATURES             Location/Qualifiers
     source          1..6391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     810..829
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(849..926)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1132..1151
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1235..1254
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1235..1253
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1254..1278
                     /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             1293..1315
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1323..1355
                     /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"
     CDS             2460..2483
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2490..2507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2560..2578)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2574..2621
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2658..3113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2745..2764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2955..2976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3205..4020)
                     /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(3205..4020)
                     /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     3928..3947
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4012..4031
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4142..4730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4631..4650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4884..4901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4916..5055
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4981..5000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5141..5163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5157..5348)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6157..6391,1..848))
                     /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"
ORIGIN
        1 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
       61 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
      121 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
      181 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
      241 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
      301 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
      361 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
      421 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
      481 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
      541 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
      601 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
      661 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
      721 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
      781 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
      841 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
      901 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
      961 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     1021 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     1081 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     1141 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     1201 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     1261 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     1321 atatggctag catgactggt ggacagcaaa tgggtcgcgg atccgaattc gagctcatgt
     1381 tttctggctt tagttctttt ggcggcggaa atggttttgt aaatatgcct caaacgtttg
     1441 aggagttctt tagatgctac cctatagcta tgatgaatga tagaatacgg aaggatgacg
     1501 caaacttcgg cgggaaaata ttcctgccac caagtgctct aagcaaacta tccatgttga
     1561 acatatgcta ccccatgctt tttaaactaa ctgccaatga aactggacgt gtgacacatg
     1621 gaggtgtttt agaatttatt gctgaagagg gcagagttta cttaccacaa tggatgatgg
     1681 aaactttggg tatacagcca ggatctctac tgcaaatttc atctactgat gtacctctgg
     1741 gccagtttgt gaagttagaa cctcagtctg tagatttctt ggatatttcc gaccccaaag
     1801 cagttttgga aaacgtactg aggaatttct cgactttgac cgtggacgat gttattgaga
     1861 ttagttacaa tggtaaaact ttcaaaatta agatcttaga agtcaaacca gaatcatcat
     1921 cgaagagtat atgtgtcatt gaaacagatt tagtaacaga ttttgctcca cctgtaggat
     1981 atgtggaacc ggattacaaa gcgctgaaag ctcagcaaga taaagaaaaa aagaattctt
     2041 ttggtaaggg ccaagttttg gatccctcgg tgcttggcca gggatcgatg tccacaagaa
     2101 tcgactatgc gggtatcgct aacagtagca gaaacaaact atctaaattt gtaggtcagg
     2161 gacaaaacat atctggtaaa gctccgaaag cggagcccaa gcaagatata aaagatatga
     2221 agataacgtt tgatggcgag cccgccaagc tagatctgcc tgaaggccaa ttattctttg
     2281 gtttcccaat ggtcctaccg aaagaagacg aagaaagtgc cgctggtagc aaatccagtg
     2341 aacaaaattt ccaaggccaa gggatctcac taaggaagag caataagaga aaaacgaaaa
     2401 gtgaccatga ttcgtccaag tccaaggctc ccaaaagccc ggaagtaatc gaaattgacc
     2461 tggaagttct gttccagggg cccctcgagc accaccacca ccaccactga gatccggctg
     2521 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     2581 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     2641 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2701 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2761 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2821 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2881 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2941 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3001 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3061 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3121 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3181 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     3241 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     3301 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     3361 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     3421 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     3481 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     3541 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     3601 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     3661 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     3721 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     3781 aggcataaat tccgtcagcc agtttagtct gaccatttca tctgtaacat cattggcaac
     3841 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     3901 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     3961 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     4021 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     4081 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4141 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4201 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4261 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4321 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4381 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4441 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4501 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4561 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4621 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4681 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4741 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4801 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4861 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4921 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4981 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5041 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5101 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5161 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5221 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5281 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5341 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5401 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5461 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5521 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5581 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5641 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5701 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5761 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5821 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5881 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     5941 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     6001 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     6061 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6121 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6181 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6241 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     6301 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6361 aaaatcctgt ttgatggtgg ttaacggcgg g
//
