LOCUS       sequence-446635-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Npl4 D460K/Y461A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7106)
  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 7106)
  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: pAM289
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     276..295
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(315..392)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     598..617
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        705..723
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    724..748
                     /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             763..785
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             804..821
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        935..953
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    954..978
                     /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             2776..2820
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2858..2876)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2872..2919
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2956..3411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3043..3062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3253..3274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3526..4317,4318..4386))
                     /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     4149..4168
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4387..4479)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4560..5148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5049..5068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5302..5319
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5334..5473
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5399..5418)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5559..5581
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5575..5766)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6338..7106,1..314))
                     /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 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
       61 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
      121 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
      181 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
      241 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
      301 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
      361 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
      421 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
      481 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
      541 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
      601 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
      661 gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat
      721 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
      781 ggagatatac catgggcagc agccatcacc atcatcacca cagccaggat ccgaattcga
      841 gctcggcgcg cctgcaggtc gacaagcttg cggccgcata atgcttaagt cgaacagaaa
      901 gtaatcgtat tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg
      961 tgagcggata acaattcccc atcttagtat attagttaag tataagaagg agatatacat
     1021 atgcttatca gatttagatc aaaaaacggt acacacaggg tttcttgtca agaaaacgac
     1081 cttttcggaa cggtcattga aaagttggtg ggtaatctgg accccaatgc cgatgtcgac
     1141 acatttactg tttgtgagaa gcccggtcaa ggtattcatg ctgtttctga actagctgat
     1201 cgaacagtga tggatctagg actaaagcac ggtgacatgc tgatccttaa ctattcggac
     1261 aagcccgcta atgagaaaga tggtgtcaat gttgaaatcg gatccgtagg tattgacagc
     1321 aagggaattc gtcaacacag gtacggtcca ctaaggatca aagaactcgc tgtagatgag
     1381 gaactagaga aagaggacgg attaattcct cgtcaaaaat caaaactatg caagcacggt
     1441 gatagaggta tgtgtgaata ctgctcgcct ttacctcctt gggacaaaga atatcatgag
     1501 aagaataaga tcaaacatat atcgtttcat tcatatctta aaaagctaaa tgaaaatgct
     1561 aataagaagg agaacggcag ctcttatatt tcccctcttt cagaacctga ttttagaatc
     1621 aataagcgtt gccataatgg ccatgaacca tggcctcggg ggatatgttc taaatgtcaa
     1681 ccatcggcaa ttacattaca acagcaagaa tttagaatgg ttgaccacgt tgaatttcag
     1741 aagagtgaaa taattaatga atttattcag gcgtggaggt acacgggtat gcaaagattt
     1801 ggctatatgt acggatctta ttctaagtat gataacacac ctttaggtat aaaggccgtc
     1861 gttgaggcga tatacgagcc ccctcagcat gatgagcaag acggtttaac catggacgta
     1921 gaacaggtca agaatgaaat gctacagatt gatagacagg ctcaagaaat ggggctttcg
     1981 cgaattggcc taatatttac agatttatct gacgcgggag ctggggatgg gtctgttttt
     2041 tgcaaaagac ataaggattc gttttttctt tcatcattag aagttattat ggctgctagg
     2101 catcaaacaa ggcatcctaa tgtaagcaag tatagcgaac aaggtttttt ttcttccaag
     2161 tttgtaactt gcgttatatc aggtaatttg gaaggtgaaa ttgatatttc gagctaccaa
     2221 gtatccacag aagctgaagc attggttact gcagatatga taagtgggtc cacatttcct
     2281 tcaatggcat atattaatga cactacagat gaaagatatg tacctgagat attttacatg
     2341 aagtcgaatg aatatggtat aacagtgaag gaaaatgcga agcctgcatt tccggtaaag
     2401 gcgcttttag tgacgctgac tcatgggttc ccgaataccg atacggaaac caactcgaaa
     2461 ttcgttagtt ccaccggatt tccatggagc aatcgacaag ctatggggca atctcaagat
     2521 tatcaagaat taaaaaagta tttattcaat gtggcttcaa gtggagattt caatcttttg
     2581 catgaaaaaa tctcgaactt tcatttacta ttatacataa attctctgca gatactctct
     2641 ccagacgaat ggaagttact aatagaatct gctgtgaaaa atgaatggga agaatctcta
     2701 ctaaaacttg tctcatcggc tggttggcaa acgttagtca tgatccttca ggaaagcggc
     2761 tagctcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg
     2821 tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa
     2881 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2941 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3001 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3061 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3121 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3181 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3241 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3301 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3361 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca
     3421 cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3481 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3541 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3601 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3661 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3721 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3781 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3841 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3901 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3961 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4021 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4081 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4141 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4201 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4261 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4321 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4381 actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga
     4441 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct
     4501 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4561 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4621 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4681 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4741 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4801 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4861 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4921 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4981 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5041 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5101 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5161 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5221 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5281 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     5341 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     5401 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     5461 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5521 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5581 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5641 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5701 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5761 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5821 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5881 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5941 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     6001 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     6061 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     6121 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     6181 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc
     6241 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     6301 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6361 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6421 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     6481 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     6541 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     6601 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     6661 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     6721 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     6781 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6841 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6901 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6961 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     7021 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     7081 gattgtgcac cgccgcttta caggct
//
