LOCUS       sequence-446629-        7820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His6-GST-3C-Npl4.
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: pAM290
FEATURES             Location/Qualifiers
     source          1..7820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1249..1272)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1279..1932)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1299..1321)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             complement(1939..1956)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    2005..2029
                     /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)."
     promoter        complement(2030..2048)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2162..2179)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2198..2220
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2235..2259
                     /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)."
     promoter        complement(2260..2278)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2366..2385)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2591..2668
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2669..3751
                     /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     complement(2688..2707)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4323..4514
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4508..4530)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4616..4755
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4671..4690
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4770..4787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5021..5040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5610..5702
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5703..5771,5772..6563)
                     /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     complement(5921..5940)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6678..7133)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6815..6836)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7027..7046
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(7170..7217)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     7213..7231
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(7269..7313)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
ORIGIN
        1 cccacttatc atatctgcag taaccaatgc ttcagcttct gtggatactt ggtagctcga
       61 aatatcaatt tcaccttcca aattacctga tataacgcaa gttacaaact tggaagaaaa
      121 aaaaccttgt tcgctatact tgcttacatt aggatgcctt gtttgatgcc tagcagccat
      181 aataacttct aatgatgaaa gaaaaaacga atccttatgt cttttgcaaa aaacagaccc
      241 atccccagct cccgcgtcag ataaatctgt aaatattagg ccaattcgcg aaagccccat
      301 ttcttgagcc tgtctatcaa tctgtagcat ttcattcttg acctgttcta cgtccatggt
      361 taaaccgtct tgctcatcat gctgaggggg ctcgtatatc gcctcaacga cggcctttat
      421 acctaaaggt gtgttatcat acttagaata agatccgtac atatagccaa atctttgcat
      481 acccgtgtac ctccacgcct gaataaattc attaattatt tcactcttct gaaattcaac
      541 gtggtcaacc attctaaatt cttgctgttg taatgtaatt gccgatggtt gacatttaga
      601 acatatcccc cgaggccatg gttcatggcc attatggcaa cgcttattga ttctaaaatc
      661 aggttctgaa agaggggaaa tataagagct gccgttctcc ttcttattag cattttcatt
      721 tagcttttta agatatgaat gaaacgatat atgtttgatc ttattcttct catgatattc
      781 tttgtcccaa ggaggtaaag gcgagcagta ttcacacata cctctatcac cgtgcttgca
      841 tagttttgat ttttgacgag gaattaatcc gtcctctttc tctagttcct catctacagc
      901 gagttctttg atccttagtg gaccgtacct gtgttgacga attcccttgc tgtcaatacc
      961 tacggatccg atttcaacat tgacaccatc tttctcatta gcgggcttgt ccgaatagtt
     1021 aaggatcagc atgtcaccgt gctttagtcc tagatccatc actgttcgat cagctagttc
     1081 agaaacagca tgaatacctt gaccgggctt ctcacaaaca gtaaatgtgt cgacatcggc
     1141 attggggtcc agattaccca ccaacttttc aatgaccgtt ccgaaaaggt cgttttcttg
     1201 acaagaaacc ctgtgtgtac cgttttttga tctaaatctg ataagcatcg gaccctggaa
     1261 gagcacctcc agatccgatt ttggaggatg gtcgccacca ccaaacgtgg cttgccagcc
     1321 ctgcaaaggc catgctatat acttgctgga tttcaagtac ttatcaattt gtgggatagc
     1381 ttcaatacgt tttttaaaac aaactaattt tgggaacgca tccaggcaca ttgggtccat
     1441 gtataaaaca acatcaagag cgtcatacaa catgaagtca ggatgggtta catgatcacc
     1501 atttaaatat gttttatgac ataaacgatc ttcgaacatt ttcagcattt caggtagctt
     1561 gctaagaaaa tcaactttga gagtttcaaa gtctttacta tatgcaattc tcgaaacacc
     1621 gtatctaata tccaaaaccg ctccttcaag cattgaaatc tctgcacgct cttttggaca
     1681 accacccaac atgttgtgct tgtcagctat ataacgtatg atggccatag actgtgttaa
     1741 tttaacatca ccatcaatat aataaggaag attgggaaac tccaaaccca attcaaactt
     1801 tttgtttcgc catttatcac cttcatcgcg ctcatacaaa tgctcttcat atttttcttc
     1861 aagatattcc aaaagaagtc gagtgggttg cacaaggccc ttaattttcc aataacctag
     1921 tataggggac attcctgagt gatgatgatg atgatggccc atatgtatat ctccttctta
     1981 tacttaacta atatactaag atggggaatt gttatccgct cacaattccc ctatagtgag
     2041 tcgtattaat ttcgattatg cggccgtgta caatacgatt actttctgtt cgacttaagc
     2101 attatgcggc cgcaagcttg tcgacctgca ggcgcgccga gctcgaattc ggatcctggc
     2161 tgtggtgatg atggtgatgg ctgctgccca tggtatatct ccttcttaaa gttaaacaaa
     2221 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     2281 ttcgcgggat cgagatcgat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc
     2341 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg
     2401 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg
     2461 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc
     2521 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt
     2581 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     2641 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     2701 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     2761 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     2821 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     2881 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     2941 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     3001 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     3061 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     3121 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     3181 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     3241 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     3301 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     3361 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     3421 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt
     3481 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     3541 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     3601 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     3661 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     3721 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     3781 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     3841 tccggtgggc gcggggcatg actagcatga tcgtgctcct gtcgttgagg acccggctag
     3901 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     3961 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     4021 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     4081 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     4141 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     4201 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     4261 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     4321 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     4381 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     4441 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     4501 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4561 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     4621 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     4681 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4741 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4801 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4861 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5581 tcacgttaag ggattttggt catgacctat ttgtttattt ttctaaatac attcaaatat
     5641 gtatccgctc atgagacaat aaccctgata aatgcttcaa tcatgattga aaaaggaaga
     5701 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5761 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5821 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5881 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5941 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6001 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6061 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6121 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6181 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6241 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6301 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6361 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6421 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6481 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6541 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6601 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6661 tgccatcgtg ccgccagaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     6721 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6781 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6841 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6901 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6961 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     7021 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     7081 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     7141 gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
     7201 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag cctaggttaa ttaagctgcg
     7261 ctagtagacg agtccatgtg ctggcgttca aatttcgcag cagcggtttc tttaccagac
     7321 tcgagctagc cgctttcctg aaggatcatg actaacgttt gccaaccagc cgatgagaca
     7381 agttttagta gagattcttc ccattcattt ttcacagcag attctattag taacttccat
     7441 tcgtctggag agagtatctg cagagaattt atgtataata gtaaatgaaa gttcgagatt
     7501 ttttcatgca aaagattgaa atctccactt gaagccacat tgaataaata cttttttaat
     7561 tcttgataat cttgagattg ccccatagct tgtcgattgc tccatggaaa tccggtggaa
     7621 ctaacgaatt tcgagttggt ttccgtatcg gtattcggga acccatgagt cagcgtcact
     7681 aaaagatagt ctaccggaaa tgcaggcttc gcattttcct tcactgttat accatattca
     7741 ttcgacttca tgtaaaatat ctcaggtaca tatctttcat ctgtagtgtc attaatatat
     7801 gccattgaag gaaatgtgga
//
