LOCUS       sequence-446595-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of cdc48 W561A/Y562A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7919)
  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 7919)
  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: pAM285
FEATURES             Location/Qualifiers
     source          1..7919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     192..211
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        299..317
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    318..342
                     /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             357..379
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             398..415
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             428..451
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        3031..3049
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3050..3074
                     /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)."
     CDS             3183..3227
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3265..3283)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3279..3326
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3363..3818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3450..3469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3660..3681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3933..4724,4725..4793))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4556..4575
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4794..4886)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4967..5555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5456..5475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5709..5726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5741..5880
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5806..5825)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5966..5988
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5982..6173)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6745..7827)
                     /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     7789..7808
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7828..7905)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
       61 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
      121 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
      181 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
      241 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
      301 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
      361 tttaacttta agaaggagat ataccatggg cagcagccat caccatcatc accacagcca
      421 ggatccgctg gaagttctgt tccaggggcc catgggtgaa gaacataaac cacttttgga
      481 cgcctcgggt gtcgaccctc gtgaggagga taaaaccgcc accgccattt taagaagaaa
      541 gaagaaggac aacatgcttt tggtggacga tgctatcaat gatgacaatt ctgtcattgc
      601 tattaattct aacaccatgg acaaattgga attgttccgt ggtgacaccg ttctcgttaa
      661 gggcaagaag agaaaagata ctgtcttaat tgtgttgatc gatgacgaat tagaagacgg
      721 agcatgcagg ataaaccgtg tagttcgtaa caatttacgt attaggctgg gtgatttagt
      781 tacaattcat ccttgccccg atatcaaata cgctactaga atttctgtgt taccaattgc
      841 tgatacgatc gaaggtataa ctggtaatct tttcgatgtt tttttgaagc cttattttgt
      901 ggaagcctac agaccagtga ggaaaggcga ccattttgtt gtcagaggcg gtatgagaca
      961 agtcgaattc aaggttgtgg atgtcgaacc tgaagaatat gccgtcgttg ctcaggatac
     1021 tattattcac tgggaaggtg agccaatcaa cagggaagat gaagaaaata atatgaatga
     1081 ggtgggttac gatgatattg gtggttgtcg taagcaaatg gctcaaatta gggaaatggt
     1141 tgaactacca ttgagacatc ctcagttgtt caaggctatc ggtatcaagc caccaagagg
     1201 tgttttgatg tatggtcccc ctggtactgg taaaaccttg atggcaagag ctgttgctaa
     1261 tgagaccggt gccttttttt tcttaattaa tggtccagaa gtcatgtcaa aaatggctgg
     1321 tgaatcagag tctaacttaa gaaaggcctt tgaagaagcc gaaaagaacg ctccagccat
     1381 catttttatc gatgaaatcg attctatagc tccaaagaga gacaagacta atggtgaagt
     1441 tgagagaaga gttgtctctc aattgttaac tttaatggat ggtatgaagg caagatctaa
     1501 tgttgttgtc attgctgcta ccaatagacc aaattcgatt gatcccgctt taagaagatt
     1561 tggtagattc gatcgtgaag tcgacattgg tatcccagat gctacaggta gacttgaagt
     1621 tctacgtatt cacaccaaga acatgaagtt ggctgatgat gttgatttgg aagcattggc
     1681 tgctgaaaca cacggttacg tgggtgctga tattgcctct ttgtgttctg aagctgccat
     1741 gcaacagatc cgtgaaaaaa tggacttgat agatttagat gaagatgaaa ttgatgcaga
     1801 ggtgctcgat tctttaggag tcaccatgga caacttcaga ttcgcattgg gcaattcaaa
     1861 cccatctgca ttgcgtgaaa ccgttgttga aagtgtcaat gttacttggg atgacgttgg
     1921 tggcctagat gaaatcaagg aggaattgaa ggaaactgta gaatacccag tcttacatcc
     1981 agaccaatac acaaagtttg ggttatcacc atcaaagggt gtgttgttct acggtccacc
     2041 aggtacaggt aaaactttgt tagcaaaagc tgtagctact gaggtttctg ccaactttat
     2101 ctcagtcaaa ggtccagaat tattgagtat ggctgcaggt gaatcggaat ctaacatccg
     2161 tgatattttt gataaggcaa gagcagctgc accaaccgtc gtttttctag atgaattaga
     2221 ctccattgcc aaggcaagag gtggttcctt gggtgatgct ggtggtgctt cggatagagt
     2281 cgtcaaccaa ctattaacag aaatggatgg tatgaatgct aagaaaaatg tgttcgtgat
     2341 tggtgccacc aataggccag atcaaattga tcctgctatt ttaagacctg gcagattgga
     2401 tcaattaatt tatgttccat tgccagatga aaacgctaga ttatctattc taaatgctca
     2461 attaaggaaa acaccactag aaccagggct agagttaact gcgattgcca aggccactca
     2521 aggtttctct ggtgctgatt tactctatat tgtgcagagg gctgctaaat acgctatcaa
     2581 ggattctatc gaagctcaca gacaacacga agctgaaaag gaagtgaaag tagaaggtga
     2641 agacgtggaa atgacagatg agggagcaaa ggctgaacaa gaaccagaag tagatcctgt
     2701 tccatatatt acaaaggagc atttcgcaga agctatgaag accgctaaac gttctgtttc
     2761 agatgctgaa ttgcgtcgtt atgaagcata ctcacagcaa atgaaagcat ctagaggtca
     2821 attcagtaat ttcaacttta atgacgctcc attaggtact acggctacgg acaatgctaa
     2881 cagtaataat agtgctccaa gtggagcagg tgctgcattt ggttctaatg cggaggaaga
     2941 tgatgatttg tatagttaga agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa
     3001 tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag
     3061 cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg
     3121 cagatctcaa ttggatatcg gccggccacg cgatcgctga cgtcggtacc ctcgagtctg
     3181 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     3241 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     3301 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     3361 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3421 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3481 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3541 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3601 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3661 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3721 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3781 taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga tggcatgaga
     3841 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3901 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3961 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4021 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4081 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4141 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4201 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4261 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4321 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4381 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4441 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4501 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4561 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4621 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4681 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4741 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4801 ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4861 tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt
     4921 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4981 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5041 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5101 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5161 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5221 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5281 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5341 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5401 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5461 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5521 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5581 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5641 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5701 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     5761 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     5821 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     5881 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5941 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6001 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     6061 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     6121 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     6181 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     6241 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     6301 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     6361 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     6421 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     6481 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     6541 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     6601 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
     6661 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     6721 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6781 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     6841 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     6901 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     6961 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     7021 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     7081 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     7141 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     7201 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     7261 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     7321 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     7381 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     7441 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     7501 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     7561 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     7621 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     7681 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     7741 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     7801 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     7861 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgc
//
