LOCUS       sequence-446778-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of cdc48 E588Q.
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: pAM284
FEATURES             Location/Qualifiers
     source          1..7919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        160..178
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    179..203
                     /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             312..356
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(394..412)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      408..455
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      492..947
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(579..598)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     789..810
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1062..1853,1854..1922))
                     /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     1685..1704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1923..2015)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2096..2684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2585..2604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2838..2855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2870..3009
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2935..2954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3095..3117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3111..3302)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3874..4956)
                     /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     4918..4937
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4957..5034)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5240..5259
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5347..5365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5366..5390
                     /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             5405..5427
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5446..5463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5476..5499
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 caatgctaac agtaataata gtgctccaag tggagcaggt gctgcatttg gttctaatgc
       61 ggaggaagat gatgatttgt atagttagaa gcttgcggcc gcataatgct taagtcgaac
      121 agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg
      181 aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata
      241 tacatatggc agatctcaat tggatatcgg ccggccacgc gatcgctgac gtcggtaccc
      301 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
      361 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
      421 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
      481 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      541 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      601 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      661 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      721 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      781 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      841 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      901 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
      961 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1921 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     1981 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     2041 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2101 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2161 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2221 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2281 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2341 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2401 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2461 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2521 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2581 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2641 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2701 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2761 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2821 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2881 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2941 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3001 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3061 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3121 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3181 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3241 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3301 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3361 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3421 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3481 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3541 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3601 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3661 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3721 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     3781 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3841 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3901 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3961 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4021 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4081 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4141 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4201 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4261 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4321 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4381 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4441 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4501 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4561 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4621 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4681 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4741 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4801 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4861 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4921 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4981 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5041 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5101 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5161 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     5221 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     5281 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     5341 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     5401 taattttgtt taactttaag aaggagatat accatgggca gcagccatca ccatcatcac
     5461 cacagccagg atccgctgga agttctgttc caggggccca tgggtgaaga acataaacca
     5521 cttttggacg cctcgggtgt cgaccctcgt gaggaggata aaaccgccac cgccatttta
     5581 agaagaaaga agaaggacaa catgcttttg gtggacgatg ctatcaatga tgacaattct
     5641 gtcattgcta ttaattctaa caccatggac aaattggaat tgttccgtgg tgacaccgtt
     5701 ctcgttaagg gcaagaagag aaaagatact gtcttaattg tgttgatcga tgacgaatta
     5761 gaagacggag catgcaggat aaaccgtgta gttcgtaaca atttacgtat taggctgggt
     5821 gatttagtta caattcatcc ttgccccgat atcaaatacg ctactagaat ttctgtgtta
     5881 ccaattgctg atacgatcga aggtataact ggtaatcttt tcgatgtttt tttgaagcct
     5941 tattttgtgg aagcctacag accagtgagg aaaggcgacc attttgttgt cagaggcggt
     6001 atgagacaag tcgaattcaa ggttgtggat gtcgaacctg aagaatatgc cgtcgttgct
     6061 caggatacta ttattcactg ggaaggtgag ccaatcaaca gggaagatga agaaaataat
     6121 atgaatgagg tgggttacga tgatattggt ggttgtcgta agcaaatggc tcaaattagg
     6181 gaaatggttg aactaccatt gagacatcct cagttgttca aggctatcgg tatcaagcca
     6241 ccaagaggtg ttttgatgta tggtccccct ggtactggta aaaccttgat ggcaagagct
     6301 gttgctaatg agaccggtgc cttttttttc ttaattaatg gtccagaagt catgtcaaaa
     6361 atggctggtg aatcagagtc taacttaaga aaggcctttg aagaagccga aaagaacgct
     6421 ccagccatca tttttatcga tgaaatcgat tctatagctc caaagagaga caagactaat
     6481 ggtgaagttg agagaagagt tgtctctcaa ttgttaactt taatggatgg tatgaaggca
     6541 agatctaatg ttgttgtcat tgctgctacc aatagaccaa attcgattga tcccgcttta
     6601 agaagatttg gtagattcga tcgtgaagtc gacattggta tcccagatgc tacaggtaga
     6661 cttgaagttc tacgtattca caccaagaac atgaagttgg ctgatgatgt tgatttggaa
     6721 gcattggctg ctgaaacaca cggttacgtg ggtgctgata ttgcctcttt gtgttctgaa
     6781 gctgccatgc aacagatccg tgaaaaaatg gacttgatag atttagatga agatgaaatt
     6841 gatgcagagg tgctcgattc tttaggagtc accatggaca acttcagatt cgcattgggc
     6901 aattcaaacc catctgcatt gcgtgaaacc gttgttgaaa gtgtcaatgt tacttgggat
     6961 gacgttggtg gcctagatga aatcaaggag gaattgaagg aaactgtaga atacccagtc
     7021 ttacatccag accaatacac aaagtttggg ttatcaccat caaagggtgt gttgttctac
     7081 ggtccaccag gtacaggtaa aactttgtta gcaaaagctg tagctactga ggtttctgcc
     7141 aactttatct cagtcaaagg tccagaatta ttgagtatgt ggtatggtga atcggaatct
     7201 aacatccgtg atatttttga taaggcaaga gcagctgcac caaccgtcgt ttttctagat
     7261 cagttagact ccattgccaa ggcaagaggt ggttccttgg gtgatgctgg tggtgcttcg
     7321 gatagagtcg tcaaccaact attaacagaa atggatggta tgaatgctaa gaaaaatgtg
     7381 ttcgtgattg gtgccaccaa taggccagat caaattgatc ctgctatttt aagacctggc
     7441 agattggatc aattaattta tgttccattg ccagatgaaa acgctagatt atctattcta
     7501 aatgctcaat taaggaaaac accactagaa ccagggctag agttaactgc gattgccaag
     7561 gccactcaag gtttctctgg tgctgattta ctctatattg tgcagagggc tgctaaatac
     7621 gctatcaagg attctatcga agctcacaga caacacgaag ctgaaaagga agtgaaagta
     7681 gaaggtgaag acgtggaaat gacagatgag ggagcaaagg ctgaacaaga accagaagta
     7741 gatcctgttc catatattac aaaggagcat ttcgcagaag ctatgaagac cgctaaacgt
     7801 tctgtttcag atgctgaatt gcgtcgttat gaagcatact cacagcaaat gaaagcatct
     7861 agaggtcaat tcagtaattt caactttaat gacgctccat taggtactac ggctacgga
//
