LOCUS       sequence-268599-        6376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AUF1p45 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6376)
  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 6376)
  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(p45)
FEATURES             Location/Qualifiers
     source          1..6376
                     /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(1311..1329)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1325..1372
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1409..1864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1496..1515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1706..1727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1956..2771)
                     /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(1956..2771)
                     /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     2679..2698
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2763..2782
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2893..3481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3382..3401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3635..3652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3667..3806
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3732..3751)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3892..3914
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3908..4099)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4908..5990)
                     /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     5952..5971
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5991..6068)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6274..6293
                     /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 atgaaggcca ttcaaactcc tccccacgac actctgaagc agcgacggca
      481 cagcgggaag aatggaaaat gtttatagga ggccttagct gggacactac aaagaaagat
      541 ctgaaggact acttttccaa atttggtgaa gttgtagact gcactctgaa gttagatcct
      601 atcacagggc gatcaagggg ttttggcttt gtgctattta aagaatcgga gagtgtagat
      661 aaggtcatgg atcaaaaaga acataaattg aatgggaagg tgattgatcc taaaagggcc
      721 aaagccatga aaacaaaaga gccggttaaa aaaatttttg ttggtggcct ttctccagat
      781 acacctgaag agaaaataag ggagtacttt ggtggttttg gtgaggtgga atccatagag
      841 ctccccatgg acaacaagac caataagagg cgtgggttct gctttattac ctttaaggaa
      901 gaagaaccag tgaagaagat aatggaaaag aaataccaca atgttggtct tagtaaatgt
      961 gaaataaaag tagccatgtc gaaggaacaa tatcagcaac agcaacagtg gggatctaga
     1021 ggaggatttg caggaagagc tcgtggaaga ggtggtggcc ccagtcaaaa ctggaaccag
     1081 ggatatagta actattggaa tcaaggctat ggcaactatg gatataacag ccaaggttac
     1141 ggtggttatg gaggatatga ctacactggt tacaacaact actatggata tggtgattat
     1201 agcaaccagc agagtggtta tgggaaggta tccaggcgag gtggtcatca aaatagctac
     1261 aaaccatact aataacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1321 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1381 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1441 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1501 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1561 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1621 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1681 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1741 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1801 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1861 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1921 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     1981 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2041 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2101 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2161 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2221 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2281 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2341 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2401 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2461 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2521 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2581 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     2641 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     2701 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     2761 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     2821 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2881 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2941 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3001 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3061 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3121 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3181 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3241 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3301 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3361 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3421 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3481 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3541 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3601 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3661 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     3721 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     3781 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     3841 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3901 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     3961 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4021 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4081 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4141 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4201 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4261 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4321 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4381 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4441 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4501 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4561 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4621 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     4681 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     4741 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     4801 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     4861 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     4921 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4981 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5041 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5101 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5161 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5221 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5281 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5341 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5401 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5461 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5521 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5581 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     5641 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     5701 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     5761 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     5821 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     5881 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     5941 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6001 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6061 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     6121 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     6181 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     6241 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     6301 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     6361 ctcgatcccg cgaaat
//
