LOCUS       sequence-446620-        5741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MC-Ub-SH3-3C-His6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5741)
  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 5741)
  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: pAM301
FEATURES             Location/Qualifiers
     source          1..5741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..158
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(178..255)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     461..480
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     564..583
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        564..582
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    583..607
                     /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             620..642
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             658..885
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1072..1095
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1096..1113
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1169..1186
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1239..1257)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1253..1300
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1337..1792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1424..1443)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1634..1655
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1884..2699)
                     /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             complement(1884..2699)
                     /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     2607..2626
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2691..2710
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2821..3409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3310..3329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3563..3580
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3595..3734
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3660..3679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3820..3842
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3836..4027)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(4836..5741,1..177))
                     /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"
ORIGIN
        1 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
       61 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
      121 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
      181 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
      241 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
      301 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
      361 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
      421 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
      481 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      541 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      601 caattcccct ctgaataatt ttgtttaact ttaagaagga gatataccca tatgtgtatg
      661 cagattttcg tcaagacttt gaccggtaaa accataacat tggaagttga atcttccgat
      721 accatcgaca acgttaagtc gaaaattcaa gacaaggaag gtatccctcc agatcaacaa
      781 agattgatct ttgccggtaa gcagctagaa gacggtagaa cgctgtctga ttacaacatt
      841 cagaaggagt ccaccttaca tcttgtgcta aggctaagag gtggtatgga agaggcggag
      901 tatgtgcgtg cgctgtttga ttttaatggt aatgacgaag aagacctgcc gttcaagaaa
      961 ggcgacatcc tgcgtatccg tgacaagccg gaggaacagt ggtggaacgc ggaggatagc
     1021 gaaggcaagc gtggtatgat tccggtgccg tacgttgaga aatatcgtcc gctggaagtt
     1081 ctgttccagg ggccccatca ccatcatcac cactaatgat gaggatccga attcgagctc
     1141 cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc
     1201 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1261 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1321 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1381 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1441 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1501 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1561 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1621 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1681 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1741 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     1801 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1861 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     1921 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1981 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     2041 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     2101 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     2161 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     2221 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     2281 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     2341 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     2401 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     2461 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2521 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2581 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2641 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2701 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     2761 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2821 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2881 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2941 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3001 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3061 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3121 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3181 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3241 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3301 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3361 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3421 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3481 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3541 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3601 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3661 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3721 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3781 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3841 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3901 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3961 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4021 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4081 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4141 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4201 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4261 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4321 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4381 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4441 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4501 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4561 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4621 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4681 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4741 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4801 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4861 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4921 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4981 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5041 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5101 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5161 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5221 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5281 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5341 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5401 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5461 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5521 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5581 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5641 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5701 agggccagac tggaggtggc aacgccaatc agcaacgact g
//
