LOCUS       sequence-443132-        7523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression AtUBA1-S and AtUBC8-S in in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7523)
  AUTHORS   Pu ZX, Wang JL, Li YY, Liang LY, Tan YT, Wang ZH, Li BL, Guo GQ, 
            Wang L, Wu L
  TITLE     A Bacterial Platform for Studying Ubiquitination Cascades Anchored 
            by SCF-Type E3 Ubiquitin Ligases.
  JOURNAL   Biomolecules. 2024 Sep 25;14(10):1209. doi: 10.3390/biom14101209.
  PUBMED    39456142
REFERENCE   2  (bases 1 to 7523)
  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"; journalName: 
            "Biomolecules."; date: "2024-09-25"; volume: "14(10):1209. doi"; 
            pages: " 10.3390/biom14101209"
COMMENT     Sequence Label: pCDFDuet-AtUBC8-S-AtUBA1-S
FEATURES             Location/Qualifiers
     source          1..7523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        45..63
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    64..88
                     /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)."
     CDS             144..161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             636..680
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     promoter        801..819
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    820..844
                     /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)."
     CDS             4169..4213
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4251..4269)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4265..4312
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4483..5274)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5275..5366)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5414..6152)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(6362..7444)
                     /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     7406..7425
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7445..7522)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
       61 aggggaattg tgagcggata acaattcccc tgtagaaata attttgttta actttaataa
      121 ggagatatac catgggcagc agccatcacc atcatcacca cagccaggga tccgaattca
      181 tggcttcgaa acggatcttg aaggagctga aggatctcca gaaagaccct ccaacctcct
      241 gcagtgcagg tccagttgct gaagacatgt ttcattggca agctacaatt atgggtcctg
      301 cagagagtcc gtattcaggc ggtgtgtttc tcgttaccat tcacttccct ccggactatc
      361 cattcaaacc accaaaggtt gcatttagga cgaaggtgtt tcaccctaat atcaacagca
      421 acggaagcat ttgccttgac attttgaaag aacaatggag ccctgccctc accatttcca
      481 aggtgttgct ctcgatatgt tcgctgttaa cagatccaaa tccagatgac cctttggtac
      541 cagagattgc acacatgtac aaaaccgaca gagccaaata cgaggctact gcaagaaact
      601 ggactcagaa gtatgccatg ggcctcgagt ctggtaaaga aaccgctgct gcgaaatttg
      661 aacgccagca catggactcg tctactagcg cagcttaaga attcgagctc ggcgcgcctg
      721 caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta
      781 cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      841 ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg cagatctcaa
      901 ttggatatcg atgcttcaca agcgagctag tgaagctaac gataagaacg ataataccat
      961 catcggttcc gatttagcat catctaagaa acgccgtatc gatttcactg aatcttcgtc
     1021 tgataagtct tcttcgattt tagcttctgg tagtagcagg ggttttcacg gcgatagcgt
     1081 cgttcagcaa atcgacatgg cttttggtaa ttcgaaccgt caggagattg atgaagatct
     1141 gcacagtcgc cagctcgccg tctatggtcg tgagactatg aggcgtctct ttgcttccaa
     1201 tgttctcatc tcggggatgc acggtcttgg tgccgagatt gccaagaatc ttatacttgc
     1261 tggtgtgaag tctgtgacct tgcatgatga aagagttgta gagctatggg acttatcaag
     1321 caactttgtt ttctcggagg atgatgttgg caagaataga gcagatgctt ctgttcagaa
     1381 gttacaggat cttaacaatg ctgtggttgt ctctagcttg accaaaagct taaacaaaga
     1441 ggatctttct ggcttccagg ttgttgtatt ctctgacata agcatggaaa gagcaatcga
     1501 atttgatgac tattgtcaca gccatcagcc tcctatcgct tttgtcaagg ctgatgtcag
     1561 gggtcttttc ggctctgtct tttgtgattt tgggcctgaa tttgcggttc ttgatgttga
     1621 tggagaggaa ccccatacag gcatcattgc gtctatctct aatgagaacc aggcatttat
     1681 ttcctgtgtc gacgatgaga ggcttgagtt tgaagatggt gacctagttg ttttctcaga
     1741 agttgagggt atgacagaat tgaatgatgg gaaaccaagg aagataaaaa gtacacggcc
     1801 atattcattc acccttgatg aggacactac taactatgga acatatgtga agggtggtat
     1861 tgtcactcaa gtgaaacagc caaagttact gaatttcaag ccattgaggg aagcgcttaa
     1921 ggatccaggg gattttctat ttagtgattt ctctaagttt gatcggcctc cactccttca
     1981 cttagcattc caggcacttg atcattttaa agctgaagct ggcagatttc ctgttgctgg
     2041 ctcagaagag gacgctcaga agcttatatc gattgccaca gccatcaata ccgggcaggg
     2101 tgatttaaag gtggagaatg ttgatcagaa gcttctaaga cacttttcct ttggagccaa
     2161 ggctgtcctg aatcctatgg ctgcaatgtt tggcggtatt gttggacagg aggttgttaa
     2221 agcttgctct ggaaaatttc atcctctctt ccagtttttc tactttgatt cagtggagtc
     2281 actcccctct gaacctgtgg actcgagtga ctttgcacca agaaacagcc ggtatgatgc
     2341 ccaaatatct gtatttgggg ccaagttcca gaagaaactt gaagatgcta aagttttcac
     2401 agtagggtct ggtgctcttg gctgtgagtt cttgaaaaat ttggctctga tgggggtttc
     2461 ttgtggaagc caagggaagc tgacagtgac agatgatgat atcattgaga agagtaacct
     2521 cagtcgtcaa tttctgttcc gtgactggaa cattggacag gctaaatcca cagttgctgc
     2581 ttcggctgct gctgttataa atcctcgctt caacattgag gccctgcaaa accgtgttgg
     2641 cgcggagact gagaatgtat ttgacgatgc cttctgggag aacttaactg ttgtcgtcaa
     2701 tgcactggat aatgttaatg cgaggctcta tgttgattcg aggtgcttgt atttccagaa
     2761 gcctctcctt gagtctggaa ctcttggtac aaagtgcaac acacagtcgg ttatcccaca
     2821 tctaactgaa aattacggtg cctcaaggga cccaccagag aaacaggccc ccatgtgtac
     2881 agtgcattcc ttcccgcata acattgatca ctgtttaact tgggctcgct ctgagtttga
     2941 gggtctgctg gagaagactc ccgctgaagt gaatgcatat ctttctagcc cagttgagta
     3001 cactaactca atgatgagtg ctggtgatgc tcaggcgagg gacacattgg agaggattgt
     3061 tgagtgcctt gaaaaggaga agtgtgagac cttccaagac tgcttaacct gggcacgact
     3121 gaggtttgag gattactttg tgaaccgtgt caagcaattg atatacacat ttcctgaaga
     3181 tgctgcgact agtaccggag ctccattctg gtctgctcca aaaagattcc ctcgtccact
     3241 ccagtactcg tcgtccgacc caagtctcct taactttatc acagcgactg ctattttacg
     3301 agcagagaca tttgggatcc cgatacctga gtggactaag aacccaaagg aagcagcaga
     3361 agctgttgac agagtgatag tcccagactt tgagccaagg caagatgcaa agattgtgac
     3421 tgatgagaaa gctaccactt taaccactgc ttcggtggac gacgctgcag tcatcgacga
     3481 tctcattgct aagatcgacc agtgtaggca taacttgtcg ccagacttca ggatgaaacc
     3541 tattcagttc gaaaaggatg atgatacaaa ctatcacatg gatgtgatag caggactagc
     3601 caacatgagg gcgagaaatt acagcatccc tgaagtggac aagctgaaag caaagttcat
     3661 agcagggaga atcatcccag ccattgcgac ttcaacagcc atggctactg ggctggtctg
     3721 cctcgagctg tacaaggtcc ttgatggagg acataaagtg gaagcttaca gaaacacatt
     3781 tgccaacctg gcacttccac tattctccat ggctgaacca cttccaccaa aggtggtgaa
     3841 gcaccgtgac atggcttgga cagtttggga cagatgggtt ctcaagggaa acccaactct
     3901 tcgtgaggtg ttgcagtggc tggaggacaa agggcttagc gcatacagca tctcttgtgg
     3961 aagctgtctt ctgttcaaca gtatgttcac aaggcacaag gagaggatgg acaagaaggt
     4021 tgtggatctc gctcgggatg ttgcgaaagt cgagttaccg ccttatcgca accatcttga
     4081 tgtagtggtg gcttgtgagg atgaagatga caatgacgtc gatattcctc tcgtctctat
     4141 ctacttcagg ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca
     4201 gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca
     4261 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     4321 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     4381 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg
     4441 gccggatctt gcggcccctc ggcttgaacg aattgttaga cattatttgc cgactacctt
     4501 ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa
     4561 gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg
     4621 ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt
     4681 tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca
     4741 gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc
     4801 aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct
     4861 tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc
     4921 aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca
     4981 cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc
     5041 tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc
     5101 aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc
     5161 cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac
     5221 gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt
     5281 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5341 tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacg ctccgggcgt
     5401 gagactgcgg cgggcgctgc ggacacatac aaagttaccc acagattccg tggataagca
     5461 ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc
     5521 cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg
     5581 aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc
     5641 gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata
     5701 cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt
     5761 atctcggctc ggtgtaggtc gttcgctcca agctgggctg taagcaagaa ctccccgttc
     5821 agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagcacg
     5881 gtaaaacgcc actggcagca gccattggta actgggagtt cgcagaggat ttgtttagct
     5941 aaacacgcgg ttgctcttga agtgtgcgcc aaagtccggc tacactggaa ggacagattt
     6001 ggttgctgtg ctctgcgaaa gccagttacc acggttaagc agttccccaa ctgacttaac
     6061 cttcgatcaa accacctccc caggtggttt tttcgtttac agggcaaaag attacgcgca
     6121 gaaaaaaagg atctcaagaa gatcctttga tcttttctac tgaaccgctc tagatttcag
     6181 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat
     6241 tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     6301 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6361 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6421 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6481 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6541 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6601 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     6661 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6721 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6781 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6841 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6901 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6961 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     7021 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     7081 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     7141 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     7201 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     7261 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     7321 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     7381 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     7441 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     7501 ttttgcgcca ttcgatggtg tcc
//
