LOCUS       sequence-378090-        7832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BHR plasmid (pRA301) expressing pruR-3XFLAG from T7 promoter for 
            high yield protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7832)
  AUTHORS   Greenwich JL, Alakavuklar MA, Fuqua C
  TITLE     An Inducible T7 Polymerase System for High-Level Protein Expression 
            in Diverse Gram-Negative Bacteria.
  JOURNAL   Microbiol Resour Announc. 2023 Feb 16;12(2):e0111922. doi: 
            10.1128/mra.01119-22. Epub 2023 Jan 16.
  PUBMED    36645284
REFERENCE   2  (bases 1 to 7832)
  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: "Microbiol 
            Resour Announc."; date: "2023-02-16"; volume: "12(2):e0111922. doi";
            pages: " 10.1128/mra.01119-22. Epub 2023 Jan 16"
COMMENT     Sequence Label: pJLG029
FEATURES             Location/Qualifiers
     source          1..7832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    754..778
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     880..899
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        880..898
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    899..923
                     /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             1501..1566
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1609..1626)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1779..1865
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1957..1984
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2072..2090)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2086..2133
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             3379..4008
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4437..5510
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      5576..5770
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(6006..6028)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6114..6254
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6169..6188
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6269..6286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6440..7028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6520..6539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7274..7832,1..233))
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 ccaggggaag ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca
       61 agccttacgg tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc
      121 actgcggagc cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg
      181 ccaactacct ctgatagttg agtcgatact tcggcgatca ccgcttcccc catgatgttt
      241 aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctcca taacatcaaa
      301 catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga ctgtacccca
      361 aaaaaacagt cataacaagc catgaaaacc gccactgcgc cgttaccacc gctgcgttcg
      421 gtcaaggttc tggaccagtt gcgtgagcgc atacgctact tgcattacag cttacgaacc
      481 gaacaggctt atgtccactg ggttcgtgcc cgaattgatc acaggcagca acgctctgtc
      541 atcgttacaa tcaacatgct accctccgcg agatcatccg tgtttcaaac ccggcagctt
      601 agttgccgtt cttccgaata gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc
      661 tctcccgctg acgccgtccc ggactgatgg gctgcctgta tcgagtggtg attttgtgcc
      721 gagctgccgg tcggggagct gttggctggc tggtggcagg atatattgtg gtgtaaacaa
      781 attgacgctt agacaactta ataacacatt gcggacgttt ttaatgtact gaattaacgc
      841 cgaattgaat tcgagctcgg tacccgggga tcctctagat aatacgactc actatagggg
      901 aattgtgagc ggataacaat tccccgagag gatccgcatg cgagcttgac tagaaggaga
      961 aaaaatatga aaaagatttg gctggcgctg gctggtttag ttttagcgtt tagcgcatcg
     1021 gcggcgcagt atgaacaggc cgggacaatt gccaaaccgc agggcaaacc gatattaacc
     1081 atatccggaa acatcaccaa cacgaatgcc gagggtgcgg cgcagtttga ccgcgacatg
     1141 ctggaagcgc tcggcatgga gacggtggag acgacgacgc cctggcatga tggccgtgtt
     1201 cgcttcgatg gtgtttcgct ggcaaagctg atggatatcg tcggcgcgaa gggcacaagc
     1261 gtcacggcgg tcgctctcaa cgattatgtc agtacgatac cgattgaaga ttttaagaaa
     1321 tttaacgtta ttctggcgat caagctggat ggcaattaca tgacagttcg cgaaaaaggc
     1381 ccgttgttcg tcatctatcc ctatgacagc gacccggagc tgcagaagca gacgtattat
     1441 tcgcggtccg catggcaagt tgccaaactg atagtggaac tcgagggtcc ggctggtctg
     1501 gactacaaag accatgacgg tgattataaa gatcatgata tcgactacaa agatgacgac
     1561 gataaatagt aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
     1621 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
     1681 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
     1741 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
     1801 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     1861 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
     1921 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
     1981 tttttgcgtt tctacaaact ctcctgcagc caagcttaat tagatccggc tgctaacaaa
     2041 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2101 ggggcctcta aacgggtctt gaggggtttt ttgtaataac cgggcaggcc atgtctgccc
     2161 gtatttcgcg taaggaaatc cattatgtac tatttacttt gatccaaccc ctccgctgct
     2221 atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca
     2281 agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt
     2341 gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca
     2401 agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga
     2461 ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca
     2521 ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg
     2581 acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca
     2641 ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg
     2701 acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg
     2761 agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg
     2821 tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga
     2881 tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga
     2941 ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg
     3001 gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac
     3061 gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac
     3121 cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagcctc ccgcgcacgt
     3181 ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg
     3241 gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt
     3301 tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca
     3361 aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc
     3421 aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg
     3481 ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa
     3541 ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc
     3601 cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg
     3661 atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc
     3721 accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa
     3781 gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag
     3841 gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac
     3901 ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc
     3961 cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta
     4021 aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca
     4081 gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc
     4141 agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca
     4201 ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa
     4261 atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc
     4321 accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc
     4381 tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga
     4441 gcggtcgcaa accatccggc ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg
     4501 agaagttgaa ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg
     4561 gtgaatcgtg gcaagcggcc gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag
     4621 ccggtgcgcc gtcgattagg aagccgccca agggcgacga gcaaccagat tttttcgttc
     4681 cgatgctcta tgacgtgggc acccgcgata gtcgcagcat catggacgtg gccgttttcc
     4741 gtctgtcgaa gcgtgaccga cgagctggcg aggtgatccg ctacgagctt ccagacgggc
     4801 acgtagaggt ttccgcaggg ccggccggca tggccagtgt gtgggattac gacctggtac
     4861 tgatggcggt ttcccatcta accgaatcca tgaaccgata ccgggaaggg aagggagaca
     4921 agcccggccg cgtgttccgt ccacacgttg cggacgtact caagttctgc cggcgagccg
     4981 atggcggaaa gcagaaagac gacctggtag aaacctgcat tcggttaaac accacgcacg
     5041 ttgccatgca gcgtacgaag aaggccaaga acggccgcct ggtgacggta tccgagggtg
     5101 aagccttgat tagccgctac aagatcgtaa agagcgaaac cgggcggccg gagtacatcg
     5161 agatcgagct agctgattgg atgtaccgcg agatcacaga aggcaagaac ccggacgtgc
     5221 tgacggttca ccccgattac tttttgatcg atcccggcat cggccgtttt ctctaccgcc
     5281 tggcacgccg cgccgcaggc aaggcagaag ccagatggtt gttcaagacg atctacgaac
     5341 gcagtggcag cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag ctgatcgggt
     5401 caaatgacct gccggagtac gatttgaagg aggaggcggg gcaggctggc ccgatcctag
     5461 tcatgcgcta ccgcaacctg atcgagggcg aagcatccgc cggttcctaa tgtacggagc
     5521 agatgctagg gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc tttcctgtgg
     5581 atagcacgta cattgggaac ccaaagccgt acattgggaa ccggaacccg tacattggga
     5641 acccaaagcc gtacattggg aaccggtcac acatgtaagt gactgatata aaagagaaaa
     5701 aaggcgattt ttccgcctaa aactctttaa aacttattaa aactcttaaa acccgcctgg
     5761 cctgtgcata actgtctggc cagcgcacag ccgaagagct gcaaaaagcg cctacccttc
     5821 ggtcgctgcg ctccctacgc cccgccgctt cgcgtcggcc tatcgcggcc gctggccgct
     5881 caaaaatggc tggcctacgg ccaggcaatc taccagggcg cggacaagcc gcgccgtcgc
     5941 cactcgaccg ccggcgccca catcaaggca ccctgcctcg cgcgtttcgg tgatgacggt
     6001 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     6061 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     6121 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     6181 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     6241 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6301 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6361 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6421 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6481 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6541 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6601 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6661 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6721 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6781 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6841 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6901 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6961 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7021 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7081 cacgttaagg gattttggtc atgcatgata tatctcccaa tttgtgtagg gcttattatg
     7141 cacgcttaaa aataataaaa gcagacttga cctgatagtt tggctgtgag caattatgtg
     7201 cttagtgcat ctaacgcttg agttaagccg cgccgcgaag cggcgtcggc ttgaacgaat
     7261 ttctagctag acattatttg ccgactacct tggtgatctc gcctttcacg tagtggacaa
     7321 attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca agataagcct
     7381 gtctagcttc aagtatgacg ggctgatact gggccggcag gcgctccatt gcccagtcgg
     7441 cagcgacatc cttcggcgcg attttgccgg ttactgcgct gtaccaaatg cgggacaacg
     7501 taagcactac atttcgctca tcgccagccc agtcgggcgg cgagttccat agcgttaagg
     7561 tttcatttag cgcctcaaat agatcctgtt caggaaccgg atcaaagagt tcctccgccg
     7621 ctggacctac caaggcaacg ctatgttctc ttgcttttgt cagcaagata gccagatcaa
     7681 tgtcgatcgt ggctggctcg aagatacctg caagaatgtc attgcgctgc cattctccaa
     7741 attgcagttc gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc acaacaatgg
     7801 tgacttctac agcgcggaga atctcgctct ct
//
