LOCUS       sequence-449331-        5927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MC-Ub-UbK48R-SH3-3C-His6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  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 5927)
  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: pAM302
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(59..514)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(196..217)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     408..427
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(551..598)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     594..612
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(665..682)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(692..709)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(710..733)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1148..1375)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     RBS             1389..1411
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1426..1450
                     /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(1450..1469)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1451..1469)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1553..1572)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1778..1855
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1856..2938
                     /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(1875..1894)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3747..3938
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3932..3954)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4040..4182
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4095..4114
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4197..4214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4368..4956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4448..4467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5067..5086)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5078..5893
                     /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             5078..5893
                     /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(5151..5170)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa
       61 attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
      121 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      181 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
      241 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
      301 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
      361 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
      421 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
      481 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc
      541 ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt
      601 attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat
      661 ctcagtggtg gtggtggtgg tgctcgagtt agtggtgatg atggtgatgg ggcccctgga
      721 acagaacttc cagcggacga tatttctcaa cgtacggcac cggaatcata ccacgcttgc
      781 cttcgctatc ctccgcgttc caccactgtt cctccggctt gtcacggata cgcaggatgt
      841 cgcctttctt gaacggcagg tcttcttcgt cattaccatt aaaatcaaac agcgcacgca
      901 catactccgc ctcttccatg ccgccacgca gacgcagaac caggtgcagg gtgctttcct
      961 tttgaatgtt gtagtcactc agggtacggc cgtcctccag ctgacggccc gcgaaaatca
     1021 gacgctgttg atccggcggg atgccttctt tgtcctggat tttgctcttc acgttgtcaa
     1081 tggtgtcgct agactccacc tccagtgtaa ttgtcttacc ggttaaagtc ttcacgaaga
     1141 tttgcatgcc accacgtaat cgtaaaacta aatgcagagt agactctttc tggatattat
     1201 agtcgctcag ggtacgacca tcttccagct gcttgcccgc aaaaatcagg cgctgctggt
     1261 ccggcggaat gccttcctta tcctgaatct tgctcttcac attgtcaatg gtatcgctag
     1321 attccacttc cagggtaata gtcttgccgg tcagggtctt cacgaaaatt tgcatacaca
     1381 tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc
     1441 tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac
     1501 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
     1561 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
     1621 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
     1681 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
     1741 atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac
     1801 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     1861 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     1921 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     1981 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     2041 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     2101 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     2161 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     2221 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     2281 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     2341 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     2401 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     2461 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     2521 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     2581 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     2641 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     2701 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     2761 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     2821 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     2881 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     2941 gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct
     3001 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     3061 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
     3121 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     3181 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     3241 tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc
     3301 tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat
     3361 caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa
     3421 caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc
     3481 cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac
     3541 ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc
     3601 gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat
     3661 cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca
     3721 gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg
     3781 cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg
     3841 atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct
     3901 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     3961 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     4021 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     4081 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt
     4141 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     4201 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4261 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4321 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4381 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4441 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4501 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4561 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4621 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4681 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4741 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4801 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4861 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4921 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4981 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa
     5041 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     5101 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     5161 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     5221 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     5281 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     5341 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     5401 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     5461 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     5521 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     5581 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     5641 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     5701 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     5761 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     5821 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     5881 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgt
//
