LOCUS       sequence-443122-        5772 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression MBP-AtIAA6-HA in in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5772)
  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 5772)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-AtIAA6-HA
FEATURES             Location/Qualifiers
     source          1..5772
                     /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             2165..2191
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2192..2218
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2271..2289)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2285..2332
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2553..3212)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3127..3146
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3213..3315)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3707..3724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3841..4386)
                     /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(3921..3940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4611..5693)
                     /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     5655..5674
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5694..5771)
                     /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 cctatggcaa aggaaggtct agcactcgag atcacagagc ttcgattggg
     1621 tcttccagga gataattata gcgaaatatc agtatgcgga tcgagtaaga agaagaagag
     1681 ggtgctctcg gatatgatga cctcatcagc gttagatact gagaatgaaa acagcgtcgt
     1741 ttcatcagtt gaagatgaat cactgccggt tgtgaagagt caagcggtgg gatggccacc
     1801 tgtgtgttct tacaggagaa agaagaacaa tgaggaagca tcgaaagcta taggctacgt
     1861 gaaagtgagc atggatggtg tgccatacat gaggaagatt gaccttggtt cgagcaacag
     1921 ttatattaat ctagtcacgg ttcttgagaa tctcttcggc tgtcttggca taggagtggc
     1981 gaaggagggt aagaagtgtg aatacattat tatatacgaa gacaaggata gagactggat
     2041 gctcgtcgga gatgtacctt ggcagatgtt taaagaatca tgcaagaggc tgaggatcgt
     2101 gaagagatca gatgcaactg gttttggtct ccagcaagat aggcctctcg gcgcgcctgc
     2161 aggttaccca tacgacgtac cagattacgc ttacccatac gacgtaccag attacgctcg
     2221 acaagcttgc ggccgcataa tgcttaagta attaacctag gctgctgtca ccgctgagca
     2281 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     2341 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     2401 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt
     2461 gctttcgaat ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc
     2521 gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag
     2581 tactgttgta attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga
     2641 acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatagtg
     2701 aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc
     2761 acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc
     2821 aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg
     2881 tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa
     2941 caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat acggaactcc
     3001 ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta
     3061 tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta
     3121 cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca
     3181 acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc
     3241 gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct
     3301 cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca
     3361 acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg
     3421 gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt agtgtatgat ggtgtttttg
     3481 aggtgctcca gtggcttctg tttctatcag ctgtccctcc tgttcagcta ctgacggggt
     3541 ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag cggagtgtat actggcttac
     3601 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg
     3661 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact
     3721 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     3781 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     3841 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     3901 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg
     3961 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     4021 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     4081 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4141 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     4201 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     4261 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     4321 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     4381 tctcaagaag atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc
     4441 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag
     4501 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4561 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4621 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4681 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4741 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4801 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4861 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     4921 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4981 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5041 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5101 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5161 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5221 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5281 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5341 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5401 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5461 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5521 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5581 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5641 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5701 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5761 tcgatggtgt cc
//
