LOCUS       sequence-268601-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AUF1p37 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6172)
  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 6172)
  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(p37)
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             151..198
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     regulatory      290..299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1107..1125)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1121..1168
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1205..1660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1292..1311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1502..1523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1752..2567)
                     /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(1752..2567)
                     /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     2475..2494
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2559..2578
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2689..3277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3178..3197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3431..3448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3463..3602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3528..3547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3688..3710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3704..3895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4704..5786)
                     /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     5748..5767
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5787..5864)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6070..6089
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgcctcgtgg tagccatatg gactataagg atgacgacga taaggattac
      181 aaagacgatg atgacaaagg gtccatgtcg gaggagcagt tcggcgggga cggggcggcg
      241 gcagcggcaa cggcggcggt aggcggctcg gcgggcgagc aggagggagc catggtggcg
      301 gcgacacagg gggcagcggc ggcggcggga agcggagccg ggaccggggg cggaaccgcg
      361 tctggaggca ccgaaggggg cagcgccgag tcggaggggg cgaagattga cgccagtaag
      421 aacgaggagg atgaagggaa aatgtttata ggaggcctta gctgggacac tacaaagaaa
      481 gatctgaagg actacttttc caaatttggt gaagttgtag actgcactct gaagttagat
      541 cctatcacag ggcgatcaag gggttttggc tttgtgctat ttaaagaatc ggagagtgta
      601 gataaggtca tggatcaaaa agaacataaa ttgaatggga aggtgattga tcctaaaagg
      661 gccaaagcca tgaaaacaaa agagccggtt aaaaaaattt ttgttggtgg cctttctcca
      721 gatacacctg aagagaaaat aagggagtac tttggtggtt ttggtgaggt ggaatccata
      781 gagctcccca tggacaacaa gaccaataag aggcgtgggt tctgctttat tacctttaag
      841 gaagaagaac cagtgaagaa gataatggaa aagaaatacc acaatgttgg tcttagtaaa
      901 tgtgaaataa aagtagccat gtcgaaggaa caatatcagc aacagcaaca gtggggatct
      961 agaggaggat ttgcaggaag agctcgtgga agaggtggtg accagcagag tggttatggg
     1021 aaggtatcca ggcgaggtgg tcatcaaaat agctacaaac catactaata acaaagcccg
     1081 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1141 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1201 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1261 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1321 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1381 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1441 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1501 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1561 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1621 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1681 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1741 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1801 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1861 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1921 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1981 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2041 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2101 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2161 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2221 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2281 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2341 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2401 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2461 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2521 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2581 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     2641 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2701 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2761 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2821 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2881 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2941 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3001 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3061 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3121 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3181 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3241 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3301 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3361 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3421 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3541 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3601 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3661 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3721 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3781 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3841 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3901 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3961 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4021 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4081 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4141 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4201 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4261 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4321 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4381 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4441 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4501 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4561 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4621 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4681 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4801 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4861 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4921 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4981 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5041 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5101 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5161 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5221 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5281 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5341 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5401 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5461 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5521 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5581 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5641 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5701 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5761 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5821 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5881 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5941 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6001 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6061 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6121 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
