LOCUS       sequence-446594-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of cdc48.
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: pAM282
FEATURES             Location/Qualifiers
     source          1..7919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1974..1992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1993..2017
                     /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             2126..2170
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2208..2226)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2222..2269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2306..2761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2393..2412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2603..2624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2876..3667,3668..3736))
                     /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     3499..3518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3737..3829)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3910..4498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4399..4418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4652..4669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4684..4823
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4749..4768)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4909..4931
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4925..5116)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5688..6770)
                     /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     6732..6751
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6771..6848)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7054..7073
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7161..7179
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7180..7204
                     /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             7219..7241
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7260..7277
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7290..7313
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 gatgaagaaa ataatatgaa tgaggtgggt tacgatgata ttggtggttg tcgtaagcaa
       61 atggctcaaa ttagggaaat ggttgaacta ccattgagac atcctcagtt gttcaaggct
      121 atcggtatca agccaccaag aggtgttttg atgtatggtc cccctggtac tggtaaaacc
      181 ttgatggcaa gagctgttgc taatgagacc ggtgcctttt ttttcttaat taatggtcca
      241 gaagtcatgt caaaaatggc tggtgaatca gagtctaact taagaaaggc ctttgaagaa
      301 gccgaaaaga acgctccagc catcattttt atcgatgaaa tcgattctat agctccaaag
      361 agagacaaga ctaatggtga agttgagaga agagttgtct ctcaattgtt aactttaatg
      421 gatggtatga aggcaagatc taatgttgtt gtcattgctg ctaccaatag accaaattcg
      481 attgatcccg ctttaagaag atttggtaga ttcgatcgtg aagtcgacat tggtatccca
      541 gatgctacag gtagacttga agttctacgt attcacacca agaacatgaa gttggctgat
      601 gatgttgatt tggaagcatt ggctgctgaa acacacggtt acgtgggtgc tgatattgcc
      661 tctttgtgtt ctgaagctgc catgcaacag atccgtgaaa aaatggactt gatagattta
      721 gatgaagatg aaattgatgc agaggtgctc gattctttag gagtcaccat ggacaacttc
      781 agattcgcat tgggcaattc aaacccatct gcattgcgtg aaaccgttgt tgaaagtgtc
      841 aatgttactt gggatgacgt tggtggccta gatgaaatca aggaggaatt gaaggaaact
      901 gtagaatacc cagtcttaca tccagaccaa tacacaaagt ttgggttatc accatcaaag
      961 ggtgtgttgt tctacggtcc accaggtaca ggtaaaactt tgttagcaaa agctgtagct
     1021 actgaggttt ctgccaactt tatctcagtc aaaggtccag aattattgag tatgtggtat
     1081 ggtgaatcgg aatctaacat ccgtgatatt tttgataagg caagagcagc tgcaccaacc
     1141 gtcgtttttc tagatgaatt agactccatt gccaaggcaa gaggtggttc cttgggtgat
     1201 gctggtggtg cttcggatag agtcgtcaac caactattaa cagaaatgga tggtatgaat
     1261 gctaagaaaa atgtgttcgt gattggtgcc accaataggc cagatcaaat tgatcctgct
     1321 attttaagac ctggcagatt ggatcaatta atttatgttc cattgccaga tgaaaacgct
     1381 agattatcta ttctaaatgc tcaattaagg aaaacaccac tagaaccagg gctagagtta
     1441 actgcgattg ccaaggccac tcaaggtttc tctggtgctg atttactcta tattgtgcag
     1501 agggctgcta aatacgctat caaggattct atcgaagctc acagacaaca cgaagctgaa
     1561 aaggaagtga aagtagaagg tgaagacgtg gaaatgacag atgagggagc aaaggctgaa
     1621 caagaaccag aagtagatcc tgttccatat attacaaagg agcatttcgc agaagctatg
     1681 aagaccgcta aacgttctgt ttcagatgct gaattgcgtc gttatgaagc atactcacag
     1741 caaatgaaag catctagagg tcaattcagt aatttcaact ttaatgacgc tccattaggt
     1801 actacggcta cggacaatgc taacagtaat aatagtgctc caagtggagc aggtgctgca
     1861 tttggttcta atgcggagga agatgatgat ttgtatagtt agaagcttgc ggccgcataa
     1921 tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga aattaatacg
     1981 actcactata ggggaattgt gagcggataa caattcccca tcttagtata ttagttaagt
     2041 ataagaagga gatatacata tggcagatct caattggata tcggccggcc acgcgatcgc
     2101 tgacgtcggt accctcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca
     2161 catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata
     2221 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2281 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2341 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2401 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2461 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2521 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2581 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2641 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2701 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     2761 tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2821 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2881 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2941 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3001 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3061 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3121 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3181 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3241 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3301 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3361 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3421 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3481 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3541 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3601 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3661 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3721 aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat
     3781 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac
     3841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4021 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4621 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4681 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     4741 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     4801 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4861 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4921 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4981 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5041 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5101 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5161 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5221 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5281 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5341 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5401 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5461 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5521 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca
     5581 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5641 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5701 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5761 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5821 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5881 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5941 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     6001 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     6061 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     6121 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     6181 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     6241 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     6301 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     6361 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6421 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6481 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6541 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6601 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6661 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6721 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6781 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6841 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     6901 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     6961 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     7021 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     7081 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     7141 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     7201 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
     7261 atcaccatca tcaccacagc caggatccgc tggaagttct gttccagggg cccatgggtg
     7321 aagaacataa accacttttg gacgcctcgg gtgtcgaccc tcgtgaggag gataaaaccg
     7381 ccaccgccat tttaagaaga aagaagaagg acaacatgct tttggtggac gatgctatca
     7441 atgatgacaa ttctgtcatt gctattaatt ctaacaccat ggacaaattg gaattgttcc
     7501 gtggtgacac cgttctcgtt aagggcaaga agagaaaaga tactgtctta attgtgttga
     7561 tcgatgacga attagaagac ggagcatgca ggataaaccg tgtagttcgt aacaatttac
     7621 gtattaggct gggtgattta gttacaattc atccttgccc cgatatcaaa tacgctacta
     7681 gaatttctgt gttaccaatt gctgatacga tcgaaggtat aactggtaat cttttcgatg
     7741 tttttttgaa gccttatttt gtggaagcct acagaccagt gaggaaaggc gaccattttg
     7801 ttgtcagagg cggtatgaga caagtcgaat tcaaggttgt ggatgtcgaa cctgaagaat
     7861 atgccgtcgt tgctcaggat actattattc actgggaagg tgagccaatc aacagggaa
//
