LOCUS       sequence-378089-        7563 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BHR plasmid (pRA301) expressing mirA-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 7563)
  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 7563)
  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: pMAT36
FEATURES             Location/Qualifiers
     source          1..7563
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    741..765
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     867..886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        867..885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    886..910
                     /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             925..947
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1202..1267
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1310..1327)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1480..1566
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1658..1685
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             1720..1737
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1790..1808)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1804..1851
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             3097..3726
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4155..5228
                     /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      5294..5488
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(5724..5746)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5832..5972
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5887..5906
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5987..6004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6158..6746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6238..6257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6992..7563,1..220))
                     /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 aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca
       61 ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact gcggagccgt
      121 acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg
      181 atagttgagt cgatacttcg gcgatcaccg cttcccccat gatgtttaac tttgttttag
      241 ggcgactgcc ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg
      301 cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg taccccaaaa aaacagtcat
      361 aacaagccat gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg
      421 accagttgcg tgagcgcata cgctacttgc attacagctt acgaaccgaa caggcttatg
      481 tccactgggt tcgtgcccga attgatcaca ggcagcaacg ctctgtcatc gttacaatca
      541 acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt
      601 ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg
      661 ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg
      721 gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga
      781 caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga attgaattcg
      841 agctcggtac ccggggatcc tctagataat acgactcact ataggggaat tgtgagcgga
      901 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatgcgggc
      961 cgcaattaat tccccgtcgc tttcgatcga caccatggac tatcaggccg agtgccaatt
     1021 cgcactggag ccttccattc aagggctgat cgaaaaagcg gaacatgccg gctggaaccg
     1081 ccagcaggcc gcccttgcca tcgtcgcgct cgccagcgag catctcaccg atctcctgac
     1141 cgccggcggc ctccccgcgc cggaccaacg ccccctctcc ctcgagggtc cggctggtct
     1201 ggactacaaa gaccatgacg gtgattataa agatcatgat atcgactaca aagatgacga
     1261 cgataaatag taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     1321 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     1381 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     1441 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     1501 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     1561 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     1621 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     1681 ctttttgcgt ttctacaaac tcttgcggcc gcactcgagc accaccacca ccaccactga
     1741 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1801 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gtaataaccg
     1861 ggcaggccat gtctgcccgt atttcgcgta aggaaatcca ttatgtacta tttactttga
     1921 tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg
     1981 aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg cccttttcct
     2041 ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac tagaaccgga
     2101 gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc cgcgtcagca
     2161 ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc tgcaccaagc
     2221 tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg
     2281 accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg gcccgcagca
     2341 cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc
     2401 tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg accgtgttcg
     2461 ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc gggcgcgagg
     2521 ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg gcacagatcg
     2581 cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc
     2641 ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca
     2701 ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc gacgccctgg
     2761 cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg acggccagga
     2821 cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg ggtacgtgtt
     2881 cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg gtttgtctga
     2941 tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc gccgccgtct
     3001 aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat
     3061 gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc tgtacttaac
     3121 cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc cctgcaactc
     3181 gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg cgattgggcg
     3241 gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac gattgaccgc
     3301 gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg
     3361 gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt gcagccaagc
     3421 ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg cattgaggtc
     3481 acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc
     3541 ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga gtcccgtatc
     3601 acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct tgaatcagaa
     3661 cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa atcaaaactc
     3721 atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta agtgccggcc
     3781 gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac acgccagcca
     3841 tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag atgtacgcgg
     3901 tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag ctaccagagt
     3961 aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg cggcatggaa
     4021 aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt
     4081 tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg aacccccaag
     4141 cccgaggaat cggcgtgagc ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc
     4201 tgggtgatga cctggtggag aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg
     4261 aggcagaagc acgccccggt gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat
     4321 cccggcaacc gccggcagcc ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc
     4381 aaccagattt tttcgttccg atgctctatg acgtgggcac ccgcgatagt cgcagcatca
     4441 tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct
     4501 acgagcttcc agacgggcac gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt
     4561 gggattacga cctggtactg atggcggttt cccatctaac cgaatccatg aaccgatacc
     4621 gggaagggaa gggagacaag cccggccgcg tgttccgtcc acacgttgcg gacgtactca
     4681 agttctgccg gcgagccgat ggcggaaagc agaaagacga cctggtagaa acctgcattc
     4741 ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg
     4801 tgacggtatc cgagggtgaa gccttgatta gccgctacaa gatcgtaaag agcgaaaccg
     4861 ggcggccgga gtacatcgag atcgagctag ctgattggat gtaccgcgag atcacagaag
     4921 gcaagaaccc ggacgtgctg acggttcacc ccgattactt tttgatcgat cccggcatcg
     4981 gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt
     5041 tcaagacgat ctacgaacgc agtggcagcg ccggagagtt caagaagttc tgtttcaccg
     5101 tgcgcaagct gatcgggtca aatgacctgc cggagtacga tttgaaggag gaggcggggc
     5161 aggctggccc gatcctagtc atgcgctacc gcaacctgat cgagggcgaa gcatccgccg
     5221 gttcctaatg tacggagcag atgctagggc aaattgccct agcaggggaa aaaggtcgaa
     5281 aaggtctctt tcctgtggat agcacgtaca ttgggaaccc aaagccgtac attgggaacc
     5341 ggaacccgta cattgggaac ccaaagccgt acattgggaa ccggtcacac atgtaagtga
     5401 ctgatataaa agagaaaaaa ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa
     5461 ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc gaagagctgc
     5521 aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta
     5581 tcgcggccgc tggccgctca aaaatggctg gcctacggcc aggcaatcta ccagggcgcg
     5641 gacaagccgc gccgtcgcca ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg
     5701 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     5761 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     5821 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     5881 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     5941 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     6001 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6061 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6121 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6181 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6241 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6301 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6361 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6421 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6481 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6541 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6601 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6661 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6721 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6781 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gcatgatata tctcccaatt
     6841 tgtgtagggc ttattatgca cgcttaaaaa taataaaagc agacttgacc tgatagtttg
     6901 gctgtgagca attatgtgct tagtgcatct aacgcttgag ttaagccgcg ccgcgaagcg
     6961 gcgtcggctt gaacgaattt ctagctagac attatttgcc gactaccttg gtgatctcgc
     7021 ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt
     7081 cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc
     7141 gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt
     7201 accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg
     7261 agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat
     7321 caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca
     7381 gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat
     7441 tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt
     7501 cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag
     7561 ccg
//
