LOCUS       sequence-269662-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AUF1p40 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6229)
  AUTHORS   Poganik JR, Long MJC, Disare MT, Liu X, Chang SH, Hla T, Aye Y
  TITLE     Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding 
            proteins HuR and AUF1.
  JOURNAL   FASEB J. 2019 Nov 1:fj201901930R. doi: 10.1096/fj.201901930R.
  PUBMED    31665914
REFERENCE   2  (bases 1 to 6229)
  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: "FASEB J. 
            2019 Nov 1:fj201901930R. doi: 10.1096/fj.201901930R."
COMMENT     Sequence Label: pET28a His6Flag2AUF1(p40)
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..28
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9..27
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    28..52
                     /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             67..89
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             108..125
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             135..152
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             159..206
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     regulatory      298..307
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1172..1190)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1186..1233
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1270..1725
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1357..1376)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1567..1588
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1817..2632)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1817..2632)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2540..2559
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2624..2643
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2754..3342
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3243..3262
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3496..3513
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3528..3667
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3593..3612)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3753..3775
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3769..3960)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4769..5851)
                     /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     5813..5832
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5852..5929)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6135..6154
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
       61 aataattttg tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc
      121 atcacagcag cggcctggtg cctcgtggta gccatatgga ctataaggat gacgacgata
      181 aggattacaa agacgatgat gacaaagggt ccatgtcgga ggagcagttc ggcggggacg
      241 gggcggcggc agcggcaacg gcggcggtag gcggctcggc gggcgagcag gagggagcca
      301 tggtggcggc gacacagggg gcagcggcgg cggcgggaag cggagccggg accgggggcg
      361 gaaccgcgtc tggaggcacc gaagggggca gcgccgagtc ggagggggcg aagattgacg
      421 ccagtaagaa cgaggaggat gaaggccatt caaactcctc cccacgacac tctgaagcag
      481 cgacggcaca gcgggaagaa tggaaaatgt ttataggagg ccttagctgg gacactacaa
      541 agaaagatct gaaggactac ttttccaaat ttggtgaagt tgtagactgc actctgaagt
      601 tagatcctat cacagggcga tcaaggggtt ttggctttgt gctatttaaa gaatcggaga
      661 gtgtagataa ggtcatggat caaaaagaac ataaattgaa tgggaaggtg attgatccta
      721 aaagggccaa agccatgaaa acaaaagagc cggttaaaaa aatttttgtt ggtggccttt
      781 ctccagatac acctgaagag aaaataaggg agtactttgg tggttttggt gaggtggaat
      841 ccatagagct ccccatggac aacaagacca ataagaggcg tgggttctgc tttattacct
      901 ttaaggaaga agaaccagtg aagaagataa tggaaaagaa ataccacaat gttggtctta
      961 gtaaatgtga aataaaagta gccatgtcga aggaacaata tcagcaacag caacagtggg
     1021 gatctagagg aggatttgca ggaagagctc gtggaagagg tggtgaccag cagagtggtt
     1081 atgggaaggt atccaggcga ggtggtcatc aaaatagcta caaaccatac taataacaaa
     1141 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1201 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1261 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1321 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1381 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1441 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1501 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1561 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1621 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1681 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1741 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1801 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1861 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1921 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1981 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2041 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2101 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2161 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2221 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2281 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2341 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2401 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2461 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2521 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2581 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     2641 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     2701 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2761 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2821 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2881 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2941 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3001 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3061 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3121 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3181 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3241 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3301 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3361 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3421 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3481 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3541 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3601 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3661 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3721 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3781 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     3841 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     3901 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3961 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4021 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4081 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4141 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4201 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4261 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4321 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4381 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     4441 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4501 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4561 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     4621 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     4681 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     4741 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4801 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4861 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4921 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4981 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5041 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5101 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5161 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5221 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5281 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5341 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     5401 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     5461 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5521 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5581 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     5641 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     5701 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     5761 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5821 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5881 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5941 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6001 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6061 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6121 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6181 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatcc
//
