LOCUS       sequence-455601-        7136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Mce3R mutant R53A in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7136)
  AUTHORS   Panagoda NT, Balazsi G, Sampson NS
  TITLE     Mycobacterium tuberculosis Mce3R TetR-like Repressor Forms an 
            Asymmetric Four-Helix Bundle and Binds a Nonpalindrome 
            Sequencedagger.
  JOURNAL   ACS Chem Biol. 2024 Dec 20;19(12):2580-2592. doi: 
            10.1021/acschembio.4c00687. Epub 2024 Nov 15.
  PUBMED    39545866
REFERENCE   2  (bases 1 to 7136)
  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"; doi: 
            "10.1021/acschembio.4c00687"; journalName: "ACS Chem Biol"; date: 
            "2024-12-20- 20"; volume: "19"; issue: "12"; pages: "2580-2592"
COMMENT     Sequence Label: 2M-T-Mce3R_R53A
FEATURES             Location/Qualifiers
     source          1..7136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1351..1369)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1365..1412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1778..1783,1784..1800,1801..1806))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1833..1851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1919..2023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2024..2092,2093..2884)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2242..2261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3055..3643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3544..3563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3797..3814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3829..3968
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3894..3913)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4054..4076
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4070..4261)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5719..5738
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5822..5841
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5822..5840
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             5929..5951
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5971..5988
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5998..7098
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     7072..7095
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             7111..7131
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 aatggcatcc gtcgcccaac ccgttaggcg ccgcccaaag gaccggaaga agcagatttt
       61 ggatcaggcc gttggactgt tcatcgaacg tggcttccat tcggtcaaat tggaggacat
      121 tgccgaggcg gccggggtga ccgcgcgcgc gttgtatgca cactacgaca acaagcaggc
      181 gttgctcgcc gaagcgatcc gaaccggcca ggatcagtac cagagcgcgc gtcgtctcac
      241 cgagggcgag acggagccga cgccgcggcc gttgaacgcc gatctggaag acctgatcgc
      301 cgcggcggtc gcctctcggg cgttgacggt gctgtggcag cgcgaggccc gctacctcaa
      361 cgaggacgac cgcacggcgg tccggcgccg catcaacgcg atcgtcgccg gcatgcgtga
      421 cagcgtgctg ctggaggtgc ccgatctgag tccacagcat tcggagttgc gggcgtgggc
      481 ggtgtccagc actttgacca gcctgggccg gcacagccta agcctgccgg gcgaggaact
      541 gaaaaagctt ctctaccagg cgtgtatggc cgcggcaagg acgcctcccg tctgcgaatt
      601 gccgccactg ccggccggtg atgccgcacg cgacgaggcc gacgtgctgt tctcccgcta
      661 cgagaccctg ctggccgcgg gcgcgcggct gttccgtgcg cagggctatc cggccgtcaa
      721 caccagcgaa atcggcaagg gagccggcat cgcgggcccg gggctgtacc gttcgttttc
      781 ttccaaacag gccatcctgg acgcgctcat ccgccgcctc gacgagtggc gctgcctgga
      841 gtgcatccga gcgctacgag cgaatcagca agcggcacaa cggttgcgcg gccttgtcca
      901 agggcacgtt cggatcagct tggacgctcc ggatctggtg gcagtgtcgg tcaccgaact
      961 gtcgcacgcc tctgtcggag tacgcgacgg ctacctgcga aatcagggcg accgcgaggc
     1021 cgtgtggatc gacctcatcg gcaagctggt acccgcgacc agtgtcgccc aggggcgact
     1081 gctggtcgcg gcggcgatta gcttcatcga agacgtcgct cgcacctggc atctcacgcg
     1141 ctacgccgga gtcgccgacg agatcagtgg cctggcgctg gcgatcctga ccagcggggc
     1201 aggtaacctc ttgcgcgcat agtaataaca ttggaagtgg ataacggatc cgcgatcgcg
     1261 gcgcgccacc tggtggccgg ccggtaccac gcgtgcgcgc tgatccggct gctaacaaag
     1321 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1381 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
     1441 cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg
     1501 agcaggactg ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga
     1561 aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa
     1621 tggacgatat cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca
     1681 tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc
     1741 tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc
     1801 tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     1861 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1921 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1981 ataaccctga taaatgcttc aataacattg aaaaaggaag agtatgagta ttcaacattt
     2041 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2101 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2161 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2221 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     2281 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2341 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2401 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2461 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2521 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2581 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2641 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2701 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2761 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2821 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2881 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2941 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3001 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3061 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3121 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3181 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3241 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3301 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3361 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3421 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3481 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3541 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3601 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3661 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3721 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3781 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3841 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3901 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3961 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4021 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4081 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4141 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4201 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4261 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4321 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4381 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4441 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4501 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4561 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4621 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4681 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     4741 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
     4801 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
     4861 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
     4921 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
     4981 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
     5041 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
     5101 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
     5161 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     5221 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     5281 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     5341 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     5401 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     5461 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5521 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5581 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5641 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5701 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5761 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     5821 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
     5881 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
     5941 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatgg ttcttctatg
     6001 aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
     6061 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
     6121 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
     6181 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
     6241 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
     6301 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
     6361 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
     6421 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
     6481 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
     6541 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
     6601 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
     6661 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
     6721 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
     6781 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
     6841 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
     6901 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact
     6961 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
     7021 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
     7081 ctgaaagacg cgcagactaa tgggatcgag gaaaacctgt acttccaatc caatgc
//
