LOCUS       sequence-475341-        8674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression AtRBX1-T7, AtSKP1-T7 and AtCUL1-S in in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8674)
  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 8674)
  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: pETDuet-AtRBX1-T7-AtSKP1-T7-AtCUL1-S
FEATURES             Location/Qualifiers
     source          1..8674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     206..225
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        313..331
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    332..356
                     /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             371..393
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             844..876
                     /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"
     CDS             1404..1436
                     /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"
     CDS             1452..1484
                     /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"
     promoter        1580..1598
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1599..1623
                     /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             3952..3996
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4034..4052)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4048..4095
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4132..4587
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4219..4238)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4429..4450
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4702..5493,5494..5562))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5325..5344
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5563..5655)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5736..6324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6225..6244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6478..6495
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6510..6649
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6575..6594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6735..6757
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6751..6942)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7514..8596)
                     /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     8558..8577
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8597..8674)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
       61 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      121 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      181 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      241 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
      301 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      361 tagaaataat tttgtttaac tttaagaagg agatatacca tggcgactct agactccgac
      421 gttaccatga ttcctgccgg agaagcctcc agcagcgtag ccgcgtcgtc ttccaacaag
      481 aaagctaagc gattcgaaat taagaagtgg agcgccgttg ctctctgggc ttgggatatc
      541 gttgttgaca actgtgcgat ctgcagaaac cacatcatgg atctttgtat cgagtgtcag
      601 gctaatcagg ccagtgccac aagtgaagag tgcactgtag cttgggagga cgaccaaaat
      661 aactgcaata agtatttctg tattcttgat tgctcaatga aagatgacca cttggagggg
      721 gtttgcaatc acgccttcca ctttcactgc atcagcagat ggctaaagac tcgtcaagtt
      781 tgtccattgg ataacagtga gtgggagttt cagaaatatg gtcacagcca ggatccgaat
      841 tcgatggcta gcatgactgg tggacagcaa atgggtccat aaccccgaaa gaaggagata
      901 tacatatgtc tgcgaagaag attgtgttga agagttccga tggtgaatct ttcgaggttg
      961 aggaggcggt ggctctcgag tcacaaacca tagcgcatat ggttgaagac gactgcgttg
     1021 acaacggagt ccctcttcct aacgtcacga gcaagatcct cgccaaggtg atcgagtatt
     1081 gcaagaggca cgtcgaggct gctgcctcta aggccgaggc cgtcgagggt gctgctacct
     1141 ccgatgacga tcttaaggcc tgggacgctg attttatgaa gatcgatcaa gctactctct
     1201 ttgaactcat tctggctgct aattacctga atatcaagaa cttgcttgat ctaacatgtc
     1261 agacagttgc ggatatgatc aaaggaaaga ctccagaaga gatccgcaca acgttcaaca
     1321 ttaagaacga cttcacacca gaggaagagg aagaggttcg cagagagaac caatgggctt
     1381 ttgaaagctc ggcgcgcctg cagatggcta gcatgactgg tggacagcaa atgggttcgg
     1441 cgcgcctgca gatggctagc atgactggtg gacagcaaat gggtagctcg gcgcgcctgc
     1501 aggtcgacaa gcttgcggcc gcataatgct taagtcgaac agaaagtaat cgtattgtac
     1561 acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     1621 tccccatctt agtatattag ttaagtataa gaaggagata tacatatggc agatctcaat
     1681 tggatatcgg ccggccacgc gatcgctgac gtcggtacca tggagcgcaa gactattgac
     1741 ttggagcaag gatgggacta tatgcagact ggaattacta agctgaaacg gattctggaa
     1801 ggattgaatg agccagcatt tgactctgag caatatatga tgctttatac gactatctac
     1861 aacatgtgca cccagaaacc tcctcatgat tactcacagc agctttatga caagtatcgg
     1921 gaagcatttg aggaatatat taactcaaca gttttgcctg cgttgaggga gaagcatgat
     1981 gagtttatgc tgcgggagct atttaaaaga tggtcaaacc ataaagtaat ggtcagatgg
     2041 ctatcccgct tcttctacta ccttgaccgt tacttcattg ctcggagatc acttccacca
     2101 ctgaatgaag ttggcctgac atgcttccgt gacctggttt ataatgagct acattctaag
     2161 gtcaaacaag ctgtaatagc tcttgttgat aaagaacggg agggcgagca gattgatagg
     2221 gccctgctga aaaacgtatt agatatctat gtagagattg gaatggggca gatggagagg
     2281 tatgaagaag attttgaaag cttcatgctt caagatactt cttcgtatta ttctcgcaag
     2341 gcatcaagct ggattcagga agattcttgc cctgattaca tgttgaagtc tgaagaatgt
     2401 ctaaagaagg agagggagag agtggctcac tacctacact caagcagtga gccaaagctg
     2461 gttgagaaag tacaacatga attgctggtt gtgtttgcaa gtcagcttct agaaaaagaa
     2521 cactcagggt gccgtgcatt gctaagagat gacaaggtgg atgatctctc caggatgtac
     2581 aggctttacc ataaaatttt gcgaggcttg gaacctgttg caaacatctt taagcagcat
     2641 gtcacagcag agggtaacgc tcttgtccaa caggccgaag acacggctac taatcaggtt
     2701 gcaaatactg ctagcgtcca ggaacaggtt cttatcagaa aagtgattga acttcatgat
     2761 aaatacatgg tatatgtcac cgagtgtttc cagaaccaca ccctcttcca taaggctttg
     2821 aaagaggcat ttgagatttt ttgtaacaaa acggttgctg gaagttcaag tgcagaacta
     2881 cttgcaacat tttgcgacaa tattctcaaa aaggggggaa gtgaaaagct gagtgatgaa
     2941 gctatcgaag atacgcttga gaaggttgtc aaattgcttg catacataag tgacaaggat
     3001 cttttcgctg agttctacag gaagaagctg gcccgtaggc tcttatttga tcgcagtgct
     3061 aatgatgatc atgagagaag tatcctgaca aagctcaagc aacaatgtgg tggacagttt
     3121 acttctaaga tggagggcat ggtgacggat ttgacactgg caagagaaaa ccaaaacagt
     3181 ttcgaggatt atctaggcag taaccctgct gcaaacccag ggattgactt gaccgtcact
     3241 gttcttacca ctggtttctg gccaagttac aaatcatttg acataaatct acccagtgaa
     3301 atgatcaagt gtgttgaagt cttcaaaggg ttttatgaaa cgaaaacgaa acacaggaag
     3361 cttacgtgga tctattcact gggaacttgt cacataaacg ggaagtttga tcaaaaggcc
     3421 atcgagttaa tagtgtctac ttaccaggct gctgtgcttc tactctttaa cacaactgac
     3481 aagttaagtt acactgagat cttggctcaa ctgaacctaa gccatgaaga tctagttagg
     3541 ttgcttcatt ccttgtcatg tgctaagtac aagatactcc ttaaggagcc aaacaccaag
     3601 actgtctccc agaatgatgc ctttgagttc aactccaaat tcaccgatag aatgcgcaga
     3661 atcaagatcc ctcttccccc agttgatgaa aggaagaaag tcgttgaaga tgtcgataaa
     3721 gacagaagat atgcaattga tgctgccatt gtcaggatca tgaagagcag gaaagtattg
     3781 ggacatcaac aacttgtttc tgagtgtgtt gagcaactta gccgaatgtt caagcctgat
     3841 atcaaagcga tcaagaagcg tatggaggat ttaataacca gagattattt ggagagggac
     3901 aaggagaatc ctaacatgtt taggtacttg gctggtaccc tcgagtctgg taaagaaacc
     3961 gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc
     4021 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     4081 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     4141 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4201 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4261 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4321 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4381 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4441 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4501 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4561 ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag
     4621 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4681 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4741 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4801 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4861 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4921 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4981 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5041 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5101 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5161 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5221 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5281 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5341 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5401 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5461 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5521 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc
     5581 atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5641 agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga
     5701 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5761 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5821 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5881 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5941 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6001 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6061 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6121 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6181 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6241 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6301 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6361 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6421 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6481 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     6541 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6601 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     6661 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6721 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6781 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     6841 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     6901 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     6961 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     7021 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     7081 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     7141 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     7201 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     7261 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     7321 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     7381 cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     7441 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     7501 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     7621 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     7681 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     7741 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     7801 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     7861 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     7921 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     7981 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     8041 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     8101 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     8161 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     8221 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     8281 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     8341 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     8401 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     8461 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     8521 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     8581 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     8641 taccgcgaaa ggttttgcgc cattcgatgg tgtc
//
