LOCUS       sequence-446596-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Npl4 W252A/R253E.
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: pAM288
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1520..1544
                     /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(1545..1563)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(1677..1694)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1713..1735
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1750..1774
                     /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(1775..1793)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1881..1900)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2106..2183
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2184..3266
                     /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(2203..2222)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3838..4029
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4023..4045)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4131..4270
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4186..4205
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4285..4302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4456..5044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4536..4555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5125..5217
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5218..5286,5287..6078)
                     /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(5436..5455)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6193..6648)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6330..6351)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6542..6561
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6685..6732)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6728..6746
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6784..6828)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
ORIGIN
        1 gtcgattgct ccatggaaat ccggtggaac taacgaattt cgagttggtt tccgtatcgg
       61 tattcgggaa cccatgagtc agcgtcacta aaagatagtc taccggaaat gcaggcttcg
      121 cattttcctt cactgttata ccatattcat tcgacttcat gtaaaatatc tcaggtacat
      181 atctttcatc tgtagtgtca ttaatatatg ccattgaagg aaatgtggac ccacttatca
      241 tatctgcagt aaccaatgct tcagcttctg tggatacttg gtagctcgaa atatcaattt
      301 caccttccaa attacctgat ataacgcaag ttacaaactt ggaagaaaaa aaaccttgtt
      361 cgctatactt gcttacatta ggatgccttg tttgatgcct agcagccata ataacttcta
      421 atgatgaaag aaaaaacgaa tccttatgtc ttttgcaaaa aacagaccca tccccagctc
      481 ccgcgtcaga taaatctgta aatattaggc caattcgcga aagccccatt tcttgagcct
      541 gtctatcaat ctgtagcatt tcattcttga cctgttctac gtccatggtt aaaccgtctt
      601 gctcatcatg ctgagggggc tcgtatatcg cctcaacgac ggcctttata cctaaaggtg
      661 tgttatcata cttagaataa gatccgtaca tatagccaaa tctttgcata cccgtgtatt
      721 ccgccgcctg aataaattca ttaattattt cactcttctg aaattcaacg tggtcaacca
      781 ttctaaattc ttgctgttgt aatgtaattg ccgatggttg acatttagaa catatccccc
      841 gaggccatgg ttcatggcca ttatggcaac gcttattgat tctaaaatca ggttctgaaa
      901 gaggggaaat ataagagctg ccgttctcct tcttattagc attttcattt agctttttaa
      961 gatatgaatg aaacgatata tgtttgatct tattcttctc atgatattct ttgtcccaag
     1021 gaggtaaagg cgagcagtat tcacacatac ctctatcacc gtgcttgcat agttttgatt
     1081 tttgacgagg aattaatccg tcctctttct ctagttcctc atctacagcg agttctttga
     1141 tccttagtgg accgtacctg tgttgacgaa ttcccttgct gtcaatacct acggatccga
     1201 tttcaacatt gacaccatct ttctcattag cgggcttgtc cgaatagtta aggatcagca
     1261 tgtcaccgtg ctttagtcct agatccatca ctgttcgatc agctagttca gaaacagcat
     1321 gaataccttg accgggcttc tcacaaacag taaatgtgtc gacatcggca ttggggtcca
     1381 gattacccac caacttttca atgaccgttc cgaaaaggtc gttttcttga caagaaaccc
     1441 tgtgtgtacc gttttttgat ctaaatctga taagcatatg tatatctcct tcttatactt
     1501 aactaatata ctaagatggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
     1561 ttaatttcga ttatgcggcc gtgtacaata cgattacttt ctgttcgact taagcattat
     1621 gcggccgcaa gcttgtcgac ctgcaggcgc gccgagctcg aattcggatc ctggctgtgg
     1681 tgatgatggt gatggctgct gcccatggta tatctccttc ttaaagttaa acaaaattat
     1741 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
     1801 gggatcgaga tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     1861 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     1921 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     1981 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     2041 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
     2101 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     2161 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     2221 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     2281 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     2341 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2401 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2461 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2521 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2581 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     2641 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     2701 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     2761 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     2821 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     2881 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     2941 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat
     3001 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     3061 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     3121 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     3181 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3241 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     3301 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3361 tgggcgcggg gcatgactag catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3421 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3481 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3541 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3601 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3661 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3721 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3781 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3841 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3901 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     3961 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     4021 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4081 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4141 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4201 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4261 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4321 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4381 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4441 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4501 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4561 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4621 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4681 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4741 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4801 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4861 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4921 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4981 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5041 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5101 ttaagggatt ttggtcatga cctatttgtt tatttttcta aatacattca aatatgtatc
     5161 cgctcatgag acaataaccc tgataaatgc ttcaatcatg attgaaaaag gaagagtatg
     5221 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5281 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5341 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5401 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5461 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5521 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5581 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5641 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5701 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5761 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5821 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5881 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5941 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6001 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6061 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6121 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgcca
     6181 tcgtgccgcc agaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt
     6241 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     6301 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     6361 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6421 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6481 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6541 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     6601 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa
     6661 tccggatata gttcctcctt tcagcaaaaa acccctcaag acccgtttag aggccccaag
     6721 gggttatgct agttattgct cagcggtggc agcagcctag gttaattaag ctgcgctagt
     6781 agacgagtcc atgtgctggc gttcaaattt cgcagcagcg gtttctttac cagactcgag
     6841 ctagccgctt tcctgaagga tcatgactaa cgtttgccaa ccagccgatg agacaagttt
     6901 tagtagagat tcttcccatt catttttcac agcagattct attagtaact tccattcgtc
     6961 tggagagagt atctgcagag aatttatgta taatagtaaa tgaaagttcg agattttttc
     7021 atgcaaaaga ttgaaatctc cacttgaagc cacattgaat aaatactttt ttaattcttg
     7081 ataatcttga gattgcccca tagctt
//
