LOCUS       sequence-443128-        5199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression Myc-tagged F-box protein and MBP-HA-tagged substrate in 
            bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5199)
  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 5199)
  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: pACYCDuet-Myc-MBP-HA
FEATURES             Location/Qualifiers
     source          1..5199
                     /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             195..224
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             225..254
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     promoter        342..360
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    361..385
                     /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             452..1552
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1526..1549
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1592..1618
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1619..1645
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1698..1716)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1712..1759
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1980..2639)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2554..2573
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2640..2742)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3134..3151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3268..3813)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3348..3367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4038..5120)
                     /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     5082..5101
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5121..5198)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
       61 aggggaattg tgagcggata acaattcccc tgtagaaata attttgttta actttaataa
      121 ggagatatac catgggcagc agcgcatcac catcatcacc aagccaggga tccgagctcg
      181 atatcactag tcctgagcag aagctcatct ccgaggagga cctcgagcag aagctcatct
      241 ccgaggagga cctcctgact gcaggtcgac aagcttgcgg ccgcataatg cttaagtcga
      301 acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg
      361 ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga
      421 tatacatatg gcagatctca attgggaatt catgaaaatc gaagaaggta aactggtaat
      481 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
      541 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
      601 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
      661 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
      721 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
      781 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
      841 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
      901 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
      961 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     1021 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     1081 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     1141 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     1201 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     1261 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     1321 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     1381 agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat
     1441 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     1501 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctgaattcga
     1561 gcccgggagg cctctcggcg cgcctgcagg ttacccatac gacgtaccag attacgctta
     1621 cccatacgac gtaccagatt acgctcgaca agcttgcggc cgcataatgc ttaagtaatt
     1681 aacctaggct gctgtcaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1741 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     1801 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     1861 ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat ccgcttatta
     1921 tcacttattc aggcgtagca accaggcgtt taagggcacc aataactgcc ttaaaaaaat
     1981 tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca
     2041 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
     2101 ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc catattggcc
     2161 acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc
     2221 tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa
     2281 tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt
     2341 tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca
     2401 ccgtctttca ttgccatacg gaactccgga tgagcattca tcaggcgggc aagaatgtga
     2461 ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata
     2521 tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
     2581 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
     2641 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
     2701 atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa
     2761 aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag
     2821 tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta
     2881 ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt ctatcagctg
     2941 tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg ccggacatca
     3001 gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg
     3061 cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga
     3121 tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg
     3181 aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg
     3241 aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca
     3301 cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc
     3361 gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt
     3421 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg
     3481 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct
     3541 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag
     3601 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa
     3661 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag
     3721 ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag
     3781 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa
     3841 tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     3901 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
     3961 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4021 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4081 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4141 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4201 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4261 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     4321 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4381 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4441 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4501 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4561 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4621 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     4681 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     4741 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     4801 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     4861 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     4921 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     4981 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5041 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5101 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5161 gccataccgc gaaaggtttt gcgccattcg atggtgtcc
//
