LOCUS       sequence-446597-        7820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His6-GST-3C-Npl4 T255A/T571A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7820)
  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 7820)
  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: paM291
FEATURES             Location/Qualifiers
     source          1..7820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     248..270
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             297..320
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2076..2120
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2158..2176)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2172..2219
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2256..2711
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2343..2362)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2553..2574
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2826..3617,3618..3686))
                     /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     3449..3468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3687..3779)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3860..4448
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4349..4368
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4602..4619
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4634..4773
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4699..4718)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4859..4881
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4875..5066)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5638..6720)
                     /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     6682..6701
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6721..6798)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7004..7023
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7111..7129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7130..7154
                     /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             7169..7191
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7210..7227
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        7341..7359
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7360..7384
                     /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             7433..7450
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(7457..7820,1..290)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
ORIGIN
        1 ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat
       61 atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg
      121 ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac
      181 gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc
      241 ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg
      301 aggtgctctt ccagggtccg atgcttatca gatttagatc aaaaaacggt acacacaggg
      361 tttcttgtca agaaaacgac cttttcggaa cggtcattga aaagttggtg ggtaatctgg
      421 accccaatgc cgatgtcgac acatttactg tttgtgagaa gcccggtcaa ggtattcatg
      481 ctgtttctga actagctgat cgaacagtga tggatctagg actaaagcac ggtgacatgc
      541 tgatccttaa ctattcggac aagcccgcta atgagaaaga tggtgtcaat gttgaaatcg
      601 gatccgtagg tattgacagc aagggaattc gtcaacacag gtacggtcca ctaaggatca
      661 aagaactcgc tgtagatgag gaactagaga aagaggacgg attaattcct cgtcaaaaat
      721 caaaactatg caagcacggt gatagaggta tgtgtgaata ctgctcgcct ttacctcctt
      781 gggacaaaga atatcatgag aagaataaga tcaaacatat atcgtttcat tcatatctta
      841 aaaagctaaa tgaaaatgct aataagaagg agaacggcag ctcttatatt tcccctcttt
      901 cagaacctga ttttagaatc aataagcgtt gccataatgg ccatgaacca tggcctcggg
      961 ggatatgttc taaatgtcaa ccatcggcaa ttacattaca acagcaagaa tttagaatgg
     1021 ttgaccacgt tgaatttcag aagagtgaaa taattaatga atttattcag gcgtggaggt
     1081 acgcgggtat gcaaagattt ggctatatgt acggatctta ttctaagtat gataacacac
     1141 ctttaggtat aaaggccgtc gttgaggcga tatacgagcc ccctcagcat gatgagcaag
     1201 acggtttaac catggacgta gaacaggtca agaatgaaat gctacagatt gatagacagg
     1261 ctcaagaaat ggggctttcg cgaattggcc taatatttac agatttatct gacgcgggag
     1321 ctggggatgg gtctgttttt tgcaaaagac ataaggattc gttttttctt tcatcattag
     1381 aagttattat ggctgctagg catcaaacaa ggcatcctaa tgtaagcaag tatagcgaac
     1441 aaggtttttt ttcttccaag tttgtaactt gcgttatatc aggtaatttg gaaggtgaaa
     1501 ttgatatttc gagctaccaa gtatccacag aagctgaagc attggttact gcagatatga
     1561 taagtgggtc cacatttcct tcaatggcat atattaatga cactacagat gaaagatatg
     1621 tacctgagat attttacatg aagtcgaatg aatatggtat aacagtgaag gaaaatgcga
     1681 agcctgcatt tccggtagac tatcttttag tgacgctgac tcatgggttc ccgaataccg
     1741 atacggaaac caactcgaaa ttcgttagtt ccaccggatt tccatggagc aatcgacaag
     1801 ctatggggca atctcaagat tatcaagaat taaaaaagta tttattcaat gtggcttcaa
     1861 gtggagattt caatcttttg catgaaaaaa tctcgaactt tcatttacta ttatacataa
     1921 attctctgca gatactctct ccagacgaat ggaagttact aatagaatct gctgtgaaaa
     1981 atgaatggga agaatctcta ctaaaacttg tctcatcggc tggttggcaa gcgttagtca
     2041 tgatccttca ggaaagcggc tagctcgagt ctggtaaaga aaccgctgct gcgaaatttg
     2101 aacgccagca catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg
     2161 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     2221 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     2281 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2341 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2401 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2461 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2521 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2581 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2641 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2701 gtttacaatt tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc
     2761 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2821 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2881 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2941 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3001 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3061 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3121 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3181 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3241 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3301 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3361 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3421 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3481 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3541 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3601 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3661 gcgacacgga aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta
     3721 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3781 aggtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3841 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3901 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3961 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4021 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4081 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4141 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4201 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4261 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4321 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4381 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4441 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4501 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4561 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4621 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4681 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4741 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4801 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4861 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4921 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4981 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5041 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     5101 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5161 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     5221 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     5281 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5341 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5401 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5461 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5521 atgctagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5581 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     5641 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5701 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5761 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5821 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5881 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     5941 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     6001 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     6061 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     6121 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     6181 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     6241 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     6301 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     6361 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     6421 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     6481 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     6541 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     6601 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6661 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6721 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6781 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     6841 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     6901 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6961 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     7021 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     7081 ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     7141 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
     7201 ggcagcagcc atcaccatca tcaccacagc caggatccga attcgagctc ggcgcgcctg
     7261 caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta
     7321 cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     7381 ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg gccatcatca
     7441 tcatcatcac tcaggaatgt cccctatact aggttattgg aaaattaagg gccttgtgca
     7501 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
     7561 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
     7621 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
     7681 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
     7741 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
     7801 tgaaactctc aaagttgatt
//
