LOCUS       sequence-446633-        5534 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of His6-GG-UbK48R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5534)
  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 5534)
  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: pAM303
FEATURES             Location/Qualifiers
     source          1..5534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     199..220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(449..1264)
                     /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(449..1264)
                     /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     1172..1191
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1256..1275
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1386..1974
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1875..1894
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2128..2145
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2160..2299
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2225..2244)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2385..2407
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2401..2592)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3401..4483)
                     /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     4445..4464
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4484..4561)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4767..4786
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4870..4889
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4870..4888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4889..4913
                     /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             4926..4948
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4961..4978
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4985..5212
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             5268..5285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5338..5356)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5352..5399
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5436..5534,1..357)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(join(5523..5534,1..8))
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
       61 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
      121 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
      181 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
      241 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
      301 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
      361 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      421 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
      481 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
      541 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
      601 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
      661 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
      721 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
      781 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
      841 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
      901 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
      961 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     1021 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     1081 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     1141 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     1201 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     1261 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     1321 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1381 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1441 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1501 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1561 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1621 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1681 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1741 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1801 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1861 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1921 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1981 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2041 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2101 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2161 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     2221 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     2281 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2341 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2401 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     2461 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     2521 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     2581 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     2641 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     2701 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     2761 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     2821 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     2881 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     2941 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     3001 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     3061 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3121 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3181 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3241 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3301 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3361 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3421 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3481 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3541 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3601 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3661 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     3721 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     3781 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     3841 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     3901 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     3961 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4021 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4081 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4141 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4201 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4261 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4321 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4381 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4441 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4501 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4561 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     4621 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     4681 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     4741 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     4801 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctga
     4921 ataattttgt ttaactttaa gaaggagata tacccatatg caccatcatc accatcacgg
     4981 tggtatgcag attttcgtca agactttgac cggtaaaacc ataacattgg aagttgaatc
     5041 ttccgatacc atcgacaacg ttaagtcgaa aattcaagac aaggaaggta tccctccaga
     5101 tcaacaaaga ttgatctttg ccggtcggca gctagaagac ggtagaacgc tgtctgatta
     5161 caacattcag aaggagtcca ccttacatct tgtgctaagg ctaagaggtg gttaatgatg
     5221 aggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     5281 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     5341 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     5401 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     5461 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5521 cgctcctttc gctt
//
