LOCUS       sequence-446632-        6391 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of Ufd1-3C-His6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6391)
  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 6391)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pAM294
FEATURES             Location/Qualifiers
     source          1..6391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(846..878)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             886..908
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    923..947
                     /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(947..966)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(948..966)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1050..1069)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1275..1352
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1353..2435
                     /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(1372..1391)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3244..3435
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3429..3451)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3537..3676
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3592..3611
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3691..3708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3862..4450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3942..3961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4561..4580)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4572..5387
                     /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             4572..5387
                     /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(4645..4664)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5479..5934)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5616..5637)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5828..5847
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5971..6018)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6014..6032
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6085..6102)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6109..6132)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 ttgggctccg ctttcggagc tttaccagat atgttttgtc cctgacctac aaatttagat
       61 agtttgtttc tgctactgtt agcgataccc gcatagtcga ttcttgtgga catcgatccc
      121 tggccaagca ccgagggatc caaaacttgg cccttaccaa aagaattctt tttttcttta
      181 tcttgctgag ctttcagcgc tttgtaatcc ggttccacat atcctacagg tggagcaaaa
      241 tctgttacta aatctgtttc aatgacacat atactcttcg atgatgattc tggtttgact
      301 tctaagatct taattttgaa agttttacca ttgtaactaa tctcaataac atcgtccacg
      361 gtcaaagtcg agaaattcct cagtacgttt tccaaaactg ctttggggtc ggaaatatcc
      421 aagaaatcta cagactgagg ttctaacttc acaaactggc ccagaggtac atcagtagat
      481 gaaatttgca gtagagatcc tggctgtata cccaaagttt ccatcatcca ttgtggtaag
      541 taaactctgc cctcttcagc aataaattct aaaacacctc catgtgtcac acgtccagtt
      601 tcattggcag ttagtttaaa aagcatgggg tatcttatgt tcaacatgga tagtttgctt
      661 agagcacttg gtggcaggaa tattttcccg ccgaagtttg cgtcatcctt ccgtattcta
      721 tcattcatca tagctatagg gtagcatcta aagaactcct caaacgtttg aggcatattt
      781 acaaaaccat ttccgccgcc aaaagaacta aagccagaaa acatgagctc gaattcggat
      841 ccgcgaccca tttgctgtcc accagtcatg ctagccatat gtatatctcc ttcttaaagt
      901 taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc
      961 gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg tggccggcat
     1021 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
     1081 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
     1141 cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt
     1201 gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga
     1261 gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc
     1321 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     1381 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     1441 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     1501 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     1561 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     1621 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     1681 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     1741 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     1801 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     1861 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     1921 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     1981 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     2041 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     2101 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg
     2161 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca
     2221 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     2281 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     2341 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     2401 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag
     2461 ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc
     2521 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     2581 atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt
     2641 cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc
     2701 gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc
     2761 gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     2821 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     2881 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     2941 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     3001 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     3061 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     3121 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     3181 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     3241 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     3301 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     3361 tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac
     3421 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     3481 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     3541 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     3601 agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag
     3661 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3721 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3781 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3841 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3901 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3961 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4021 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4081 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4141 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4201 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4261 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4321 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4381 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4441 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4501 ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata
     4561 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
     4621 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     4681 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     4741 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     4801 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     4861 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     4921 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     4981 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     5041 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     5101 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     5161 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     5221 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     5281 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     5341 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     5401 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5461 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5521 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5581 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5641 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5701 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     5761 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     5821 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     5881 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     5941 cgccaatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc
     6001 cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg
     6061 ggctttgtta gcagccggat ctcagtggtg gtggtggtgg tgctcgaggg gcccctggaa
     6121 cagaacttcc aggtcaattt cgattacttc cgggcttttg ggagccttgg acttggacga
     6181 atcatggtca cttttcgttt ttctcttatt gctcttcctt agtgagatcc cttggccttg
     6241 gaaattttgt tcactggatt tgctaccagc ggcactttct tcgtcttctt tcggtaggac
     6301 cattgggaaa ccaaagaata attggccttc aggcagatct agcttggcgg gctcgccatc
     6361 aaacgttatc ttcatatctt ttatatcttg c
//
