LOCUS       sequence-269663-        6319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AUF1p42 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6319)
  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 6319)
  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(p42)
FEATURES             Location/Qualifiers
     source          1..6319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..50
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        31..49
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    50..74
                     /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             89..111
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             130..147
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             157..174
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             181..228
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     regulatory      320..329
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1284..1302)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1298..1345
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1382..1837
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1469..1488)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1679..1700
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1929..2744)
                     /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(1929..2744)
                     /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     2652..2671
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2736..2755
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2866..3454
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3355..3374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3608..3625
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3640..3779
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3705..3724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3865..3887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3881..4072)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4881..5963)
                     /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     5925..5944
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5964..6041)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6247..6266
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
       61 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
      121 ggcagcagcc atcatcatca tcatcacagc agcggcctgg tgcctcgtgg tagccatatg
      181 gactataagg atgacgacga taaggattac aaagacgatg atgacaaagg gtccatgtcg
      241 gaggagcagt tcggcgggga cggggcggcg gcagcggcaa cggcggcggt aggcggctcg
      301 gcgggcgagc aggagggagc catggtggcg gcgacacagg gggcagcggc ggcggcggga
      361 agcggagccg ggaccggggg cggaaccgcg tctggaggca ccgaaggggg cagcgccgag
      421 tcggaggggg cgaagattga cgccagtaag aacgaggagg atgaagggaa aatgtttata
      481 ggaggcctta gctgggacac tacaaagaaa gatctgaagg actacttttc caaatttggt
      541 gaagttgtag actgcactct gaagttagat cctatcacag ggcgatcaag gggttttggc
      601 tttgtgctat ttaaagaatc ggagagtgta gataaggtca tggatcaaaa agaacataaa
      661 ttgaatggga aggtgattga tcctaaaagg gccaaagcca tgaaaacaaa agagccggtt
      721 aaaaaaattt ttgttggtgg cctttctcca gatacacctg aagagaaaat aagggagtac
      781 tttggtggtt ttggtgaggt ggaatccata gagctcccca tggacaacaa gaccaataag
      841 aggcgtgggt tctgctttat tacctttaag gaagaagaac cagtgaagaa gataatggaa
      901 aagaaatacc acaatgttgg tcttagtaaa tgtgaaataa aagtagccat gtcgaaggaa
      961 caatatcagc aacagcaaca gtggggatct agaggaggat ttgcaggaag agctcgtgga
     1021 agaggtggtg gccccagtca aaactggaac cagggatata gtaactattg gaatcaaggc
     1081 tatggcaact atggatataa cagccaaggt tacggtggtt atggaggata tgactacact
     1141 ggttacaaca actactatgg atatggtgat tatagcaacc agcagagtgg ttatgggaag
     1201 gtatccaggc gaggtggtca tcaaaatagc tacaaaccat actaataaca aagcccgaaa
     1261 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1321 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1381 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1441 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1501 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1561 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1621 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1681 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1741 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1801 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     1861 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1921 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1981 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2041 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2101 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2161 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2221 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2281 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2341 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2401 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2461 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2521 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2581 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2641 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2701 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2761 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2821 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2881 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2941 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3001 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3061 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3121 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3181 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3241 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3301 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3361 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3421 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3481 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3541 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3601 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3661 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3721 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3781 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3841 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3901 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3961 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4021 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4081 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4141 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4201 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4261 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4321 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4381 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4441 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4501 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4561 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4621 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4681 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4741 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4801 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4861 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4921 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4981 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5041 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5101 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5161 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5221 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5281 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5341 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5401 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5461 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5521 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5581 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5641 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5701 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5761 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5821 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5881 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5941 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6001 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6061 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6121 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6181 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6241 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6301 ggccacgatg cgtccggcg
//
