LOCUS       sequence-451489-        5513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCM WT ubiquitin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5513)
  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 5513)
  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: pAM298
FEATURES             Location/Qualifiers
     source          1..5513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..50)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     241..260
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(384..431)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     427..445
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(498..515)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(571..798)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     RBS             814..836
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    849..873
                     /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)."
     primer_bind     complement(873..892)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(874..892)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(976..995)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1201..1278
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1279..2361
                     /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(1298..1317)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3170..3361
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3355..3377)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3463..3602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3518..3537
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3617..3634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3788..4376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3868..3887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4487..4506)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4498..5313
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4498..5313
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4571..4590)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5405..5513,1..347))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
       61 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
      121 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
      181 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
      241 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
      301 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat
      361 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg
      421 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg
      481 ttagcagccg gatctcagtg gtggtggtgg tggtgctcga gtgcggccgc aagcttgtcg
      541 acggagctcg aattcggatc ctcatcatta accacctctt agccttagca caagatgtaa
      601 ggtggactcc ttctgaatgt tgtaatcaga cagcgttcta ccgtcttcta gctgcttacc
      661 ggcaaagatc aatctttgtt gatctggagg gataccttcc ttgtcttgaa ttttcgactt
      721 aacgttgtcg atggtatcgg aagattcaac ttccaatgtt atggttttac cggtcaaagt
      781 cttgacgaaa atctgcatac acatatgggt atatctcctt cttaaagtta aacaaaatta
      841 ttcagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
      901 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
      961 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1021 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1081 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1141 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1201 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     1261 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     1321 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     1381 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     1441 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     1501 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     1561 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     1621 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     1681 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     1741 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     1801 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     1861 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     1921 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     1981 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     2041 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     2101 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     2161 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     2221 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     2281 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2341 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     2401 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     2461 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     2521 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     2581 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     2641 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     2701 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     2761 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     2821 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     2881 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     2941 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     3001 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     3061 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     3121 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     3181 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     3241 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     3301 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3361 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3421 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     3481 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     3541 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     3601 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3661 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3721 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3781 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3841 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3901 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3961 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4021 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4081 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4141 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4201 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4261 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4321 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4381 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4441 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     4501 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     4561 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     4621 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     4681 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     4741 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     4801 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     4861 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     4921 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     4981 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     5041 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     5101 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     5161 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     5221 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     5281 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     5341 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5401 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5461 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa aga
//
