LOCUS       sequence-443118-        6987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression AtTIR1-Myc and MBP-HA in in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6987)
  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 6987)
  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-HA
FEATURES             Location/Qualifiers
     source          1..6987
                     /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             3380..3406
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3407..3433
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3486..3504)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3500..3547
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3768..4427)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4342..4361
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4428..4530)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4922..4939)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5056..5601)
                     /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(5136..5155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5826..6908)
                     /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     6870..6889
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6909..6986)
                     /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 tctcggcgcg cctgcaggtt acccatacga cgtaccagat tacgcttacc catacgacgt
     3421 accagattac gctcgacaag cttgcggccg cataatgctt aagtaattaa cctaggctgc
     3481 tgtcaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3541 ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
     3601 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
     3661 gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag
     3721 gcgtagcaac caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc
     3781 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
     3841 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
     3901 tatttgccca tagtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
     3961 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
     4021 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
     4081 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
     4141 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
     4201 gccatacgga actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
     4261 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
     4321 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
     4381 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
     4441 gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
     4501 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag
     4561 ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc
     4621 acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt
     4681 atgatggtgt ttttgaggtg ctccagtggc ttctgtttct atcagctgtc cctcctgttc
     4741 agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag
     4801 tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca
     4861 ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt
     4921 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg
     4981 aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc
     5041 cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg
     5101 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5161 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt
     5221 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca
     5281 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact
     5341 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta
     5401 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca
     5461 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag
     5521 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg
     5581 cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt
     5641 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     5701 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5761 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     5821 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5881 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     5941 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     6001 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     6061 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     6121 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     6181 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     6241 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     6301 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     6361 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     6421 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     6481 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     6541 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     6601 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     6661 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     6721 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     6781 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     6841 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     6901 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     6961 aaggttttgc gccattcgat ggtgtcc
//
