LOCUS       sequence-449333-        6275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His6-mEos3.2-UbK48R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6275)
  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 6275)
  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: pAM305
FEATURES             Location/Qualifiers
     source          1..6275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(3..50)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     46..64
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(117..134)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(190..417)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             complement(442..1119)
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     CDS             complement(1120..1143)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1156..1173)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1195..1217
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1230..1254
                     /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(1254..1273)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1255..1273)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1357..1376)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1582..1659
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1660..2742
                     /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(1679..1698)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3551..3742
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3736..3758)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3844..3983
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3899..3918
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3998..4015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4169..4757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4249..4268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4868..4887)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4879..5694
                     /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             4879..5694
                     /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(4952..4971)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5786..6241)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5923..5944)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6135..6154
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca
       61 gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg
      121 tggtggtggt ggtgctcgag tgcggccgca agcttgtcga cggagctcga attcggatcc
      181 tcatcattaa ccacctctta gccttagcac aagatgtaag gtggactcct tctgaatgtt
      241 gtaatcagac agcgttctac cgtcttctag ctgccgaccg gcaaagatca atctttgttg
      301 atctggaggg ataccttcct tgtcttgaat tttcgactta acgttgtcga tggtatcgga
      361 agattcaact tccaatgtta tggttttacc ggtcaaagtc ttgacgaaaa tctgcatacc
      421 agatccacta ccagagccgc ttcgtctggc attgtcaggc aatccagaat gagcaacagc
      481 atgctcatac agcttaacct tgttgtaatc tttgtcatgg cttaaaatct caatgcagtg
      541 gtccacaaag tgggcgcctg gtaacttgac acccttctcc ttagctttgt aagtagttct
      601 gaagtcacat cggtaatggg catttccttc aagcaacaaa gccatctcaa tatcacccgt
      661 cagcactcca tcacgcacat acattttctc agtggagggc tcccatttca gcgtcttctt
      721 ctgcataact ggaccattgg cgggaaagtt ggtaccataa aatcgaactt tattatagaa
      781 agtgtcccct tccattgtta tgtcgtttct ggcgttgcaa atgcccccgt cttcgaaagt
      841 caagcttcgt tcccacgaat accccttagg aaacgactgc ttaaaatagt cttgtatgtt
      901 gtctggatat ttggcgaata ccctgttgcc gtaatggaat gcagtggtca ggatatcaaa
      961 ggcaaaaggc agaggtccgc cctctttgac ttcaagatcc atactctgtt ttccctcaaa
     1021 aggcttgcct gtaccatctc cgtcgatcac aaagtggtgc ccgtttacgt tgccttccat
     1081 acggagtttg atcttcatgt ctggcttaat cgcactcatg ggcccctgga acagaacttc
     1141 cagcggatcc tggctgtggt gatgatggtg atggctgctg cccatatggg tatatctcct
     1201 tcttaaagtt aaacaaaatt attcagaggg gaattgttat ccgctcacaa ttcccctata
     1261 gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg
     1321 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     1381 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     1441 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg
     1501 cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata
     1561 agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca
     1621 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     1681 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     1741 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     1801 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     1861 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     1921 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     1981 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     2041 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     2101 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     2161 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     2221 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     2281 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     2341 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     2401 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     2461 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     2521 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     2581 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     2641 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     2701 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2761 aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc
     2821 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     2881 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     2941 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     3001 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     3061 cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac
     3121 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg
     3181 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt
     3241 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt
     3301 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     3361 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag
     3421 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt
     3481 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac
     3541 ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag
     3601 ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat
     3661 ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg
     3721 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3781 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3841 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     3901 gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg
     3961 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4021 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4081 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4141 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4201 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4261 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4321 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4381 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4441 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4501 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4561 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4621 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4681 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4741 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4801 acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac
     4861 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga
     4921 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     4981 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     5041 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     5101 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     5161 tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct
     5221 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     5281 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     5341 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     5401 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     5461 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     5521 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     5581 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     5641 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta
     5701 attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5761 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5821 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5881 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5941 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     6001 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     6061 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     6121 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     6181 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     6241 tcccattcgc caatccggat atagttcctc ctttc
//
