LOCUS       sequence-459912-        7560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression AtTIR1-Myc and MBP-AtIAA6-HA in in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7560)
  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 7560)
  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-AtTIR1-Myc-MBP-AtIAA6-HA
FEATURES             Location/Qualifiers
     source          1..7560
                     /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             1983..2012
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2013..2042
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     promoter        2130..2148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2149..2173
                     /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             2240..3340
                     /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     3314..3337
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3953..3979
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3980..4006
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4059..4077)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4073..4120
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4341..5000)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4915..4934
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5001..5103)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5495..5512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5629..6174)
                     /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(5709..5728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6399..7481)
                     /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     7443..7462
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7482..7559)
                     /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 tccatgcaga
      181 agcgaatagc cttgtcgttt ccagaagagg tactagagca tgtgttctcg tttattcagc
      241 tggataagga taggaactca gtctctctgg tgtgcaagtc atggtacgag atcgagcggt
      301 ggtgcaggag gaaagtcttc atcgggaact gctacgccgt gagtccagcg acggtgatta
      361 ggaggttccc gaaagtgaga tccgtggagc ttaaaggaaa acctcacttt gctgacttta
      421 atttggtacc tgacggatgg ggaggttacg tgtatccatg gattgaggcc atgtcttcgt
      481 cttacacgtg gcttgaagag ataaggctga agaggatggt ggtcaccgac gattgcttgg
      541 agctcatagc caagtctttt aagaatttta aggttcttgt gctttcttcc tgcgaaggct
      601 tctccaccga tggtctcgct gctatcgctg ccacttgcag gaatctgaaa gagcttgact
      661 tacgagagag tgatgttgac gacgttagtg gccactggct tagccatttc ccagatacat
      721 acacttcttt ggtatcactc aatatatctt gcttagcatc tgaggtcagt ttctctgctc
      781 tggaaaggct ggtgactagg tgtcccaatc tcaagtctct caagcttaac cgagctgttc
      841 cacttgaaaa attggctact ttacttcaaa gagcacctca attggaggaa ttgggcactg
      901 gtgggtacac tgcagaagtg cgaccagatg tttactctgg tttatctgta gcgctctctg
      961 ggtgcaagga attgaggtgc ttatctggat tttgggatgc tgttcctgcc tatcttccag
     1021 cagtttattc ggtttgcagt cggcttacaa ctttgaatct gagttatgca acagtccaga
     1081 gctatgatct tgtcaagctt ctttgtcaat gccctaaact gcagcgcctc tgggtgcttg
     1141 actacatcga ggatgctggt cttgaggtgc ttgcttcaac ctgcaaggac ctacgcgagc
     1201 tgagagtgtt tccgtccgag ccttttgtca tggaaccaaa tgtggcattg acggaacagg
     1261 ggcttgtctc cgtctccatg ggctgtccaa aactcgagtc ggttctctac ttctgccgtc
     1321 aaatgaccaa tgctgcattg ataaccattg ctaggaaccg tcccaacatg actcgcttcc
     1381 gtttgtgcat cattgagcca aaagccccag actatctgac tctagagcca ctggatattg
     1441 gatttggagc catagtagag cactgcaagg atctccgtcg cctctctcta tctggcctct
     1501 tgaccgacaa ggtttttgaa tacattggga catatgccaa gaagatggaa atgctctcag
     1561 tggcatttgc aggagacagt gacttaggca tgcatcatgt tttgtccggg tgcgatagct
     1621 tgaggaaact agagataagg gactgcccgt ttggagacaa ggcgcttttg gccaatgctt
     1681 caaagctgga gacaatgcga tccctttgga tgtcttcttg ttccgtgagt tttggagcct
     1741 gcaagttact aggacagaag atgccaaagc tgaatgtgga agtcatcgat gaacggggtg
     1801 caccggactc gagaccagag agctgccctg ttgagagagt cttcatatac cgaacagtgg
     1861 ctggtcctcg atttgacatg cctggcttcg tctggaacat ggaccaagac tcaacaatga
     1921 ggttttccag gcaaatcatt actactaacg gattaggatc cgagctcgat atcactagtc
     1981 ctgagcagaa gctcatctcc gaggaggacc tcgagcagaa gctcatctcc gaggaggacc
     2041 tcctgactgc aggtcgacaa gcttgcggcc gcataatgct taagtcgaac agaaagtaat
     2101 cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc
     2161 ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata tacatatggc
     2221 agatctcaat tgggaattca tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg
     2281 cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat
     2341 taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac
     2401 tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc
     2461 tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac
     2521 ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt
     2581 atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc
     2641 ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga
     2701 accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa
     2761 cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac
     2821 cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc
     2881 agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc
     2941 caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca
     3001 accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa
     3061 agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt
     3121 taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa
     3181 agatccacgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat
     3241 cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg
     3301 tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact gaattcgagc ccgggaggcc
     3361 tatggcaaag gaaggtctag cactcgagat cacagagctt cgattgggtc ttccaggaga
     3421 taattatagc gaaatatcag tatgcggatc gagtaagaag aagaagaggg tgctctcgga
     3481 tatgatgacc tcatcagcgt tagatactga gaatgaaaac agcgtcgttt catcagttga
     3541 agatgaatca ctgccggttg tgaagagtca agcggtggga tggccacctg tgtgttctta
     3601 caggagaaag aagaacaatg aggaagcatc gaaagctata ggctacgtga aagtgagcat
     3661 ggatggtgtg ccatacatga ggaagattga ccttggttcg agcaacagtt atattaatct
     3721 agtcacggtt cttgagaatc tcttcggctg tcttggcata ggagtggcga aggagggtaa
     3781 gaagtgtgaa tacattatta tatacgaaga caaggataga gactggatgc tcgtcggaga
     3841 tgtaccttgg cagatgttta aagaatcatg caagaggctg aggatcgtga agagatcaga
     3901 tgcaactggt tttggtctcc agcaagatag gcctctcggc gcgcctgcag gttacccata
     3961 cgacgtacca gattacgctt acccatacga cgtaccagat tacgctcgac aagcttgcgg
     4021 ccgcataatg cttaagtaat taacctaggc tgctgtcacc gctgagcaat aactagcata
     4081 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga
     4141 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
     4201 agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt
     4261 ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt ttaagggcac
     4321 caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
     4381 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     4441 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa aacgggggcg
     4501 aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg
     4561 gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg
     4621 taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca
     4681 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca
     4741 ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg atgagcattc
     4801 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg
     4861 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact
     4921 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat
     4981 ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa
     5041 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga
     5101 tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca
     5161 ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg
     5221 tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag gtgctccagt
     5281 ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg tgcgtaacgg
     5341 caaaagcacc gccggacatc agcgctagcg gagtgtatac tggcttacta tgttggcact
     5401 gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc
     5461 agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg ctacgctcgg
     5521 tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg
     5581 ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct
     5641 ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
     5701 aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc
     5761 tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc
     5821 tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg
     5881 ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac
     5941 atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag
     6001 tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc
     6061 cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag
     6121 gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat
     6181 catcttatta atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta
     6241 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
     6301 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     6361 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6421 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6481 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     6541 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     6601 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     6661 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     6721 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     6781 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     6841 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     6901 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     6961 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     7021 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     7081 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     7141 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     7201 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     7261 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     7321 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     7381 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     7441 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     7501 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
//
