LOCUS       sequence-450359-        7136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Mce3R mutant K262A 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_K262A
FEATURES             Location/Qualifiers
     source          1..7136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     386..405
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     489..508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        489..507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             596..618
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             638..655
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             665..1765
                     /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     1739..1762
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1778..1798
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3154..3172)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3168..3215
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3581..3586,3587..3603,3604..3609))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3636..3654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3722..3826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3827..3895,3896..4687)
                     /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(4045..4064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4858..5446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5347..5366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5600..5617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5632..5771
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5697..5716)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5857..5879
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5873..6064)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
       61 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
      121 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
      181 gcatcggtcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
      241 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      301 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagtgctc atgagcccga
      361 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      421 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      481 cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta gtgccggctc
      541 cggagagctc tttaattaag cggccgccct gcaggactcg agttctagaa ataattttgt
      601 ttaactttaa gaaggagata tacatatgaa atcttctcac catcaccatc accatggttc
      661 ttctatgaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      721 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      781 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      841 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      901 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      961 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
     1021 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
     1081 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
     1141 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
     1201 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
     1261 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     1321 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     1381 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1441 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga
     1501 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1561 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1621 cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1681 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1741 tgaagccctg aaagacgcgc agactaatgg gatcgaggaa aacctgtact tccaatccaa
     1801 tgcaatggca tccgtcgccc aacccgttag gcgccgccca acggaccgga agaagcagat
     1861 tttggatcag gccgttggac tgttcatcga acgtggcttc cattcggtca aattggagga
     1921 cattgccgag gcggccgggg tgaccgcgcg cgcgttgtat cgccactacg acaacaagca
     1981 ggcgttgctc gccgaagcga tccgaaccgg ccaggatcag taccagagcg cgcgtcgtct
     2041 caccgagggc gagacggagc cgacgccgcg gccgttgaac gccgatctgg aagacctgat
     2101 cgccgcggcg gtcgcctctc gggcgttgac ggtgctgtgg cagcgcgagg cccgctacct
     2161 caacgaggac gaccgcacgg cggtccggcg ccgcatcaac gcgatcgtcg ccggcatgcg
     2221 tgacagcgtg ctgctggagg tgcccgatct gagtccacag cattcggagt tgcgggcgtg
     2281 ggcggtgtcc agcactttga ccagcctggg ccggcacagc ctaagcctgc cgggcgagga
     2341 actgaaaaag cttctctacc aggcgtgtat ggccgcggca aggacgcctc ccgtctgcga
     2401 attgccgcca ctgccggccg gtgatgccgc acgcgacgag gccgacgtgc tgttctcccg
     2461 ctacgagacc ctgctggccg cgggcgcgcg gctgttccgt gcgcagggct atccggccgt
     2521 caacaccagc gaaatcggca agggagccgg catcgcgggc ccggggctgt accgttcgtt
     2581 ttcttccgca caggccatcc tggacgcgct catccgccgc ctcgacgagt ggcgctgcct
     2641 ggagtgcatc cgagcgctac gagcgaatca gcaagcggca caacggttgc gcggccttgt
     2701 ccaagggcac gttcggatca gcttggacgc tccggatctg gtggcagtgt cggtcaccga
     2761 actgtcgcac gcctctgtcg aagtacgcga cggctacctg cgaaatcagg gcgaccgcga
     2821 ggccgtgtgg atcgacctca tcggcaagct ggtacccgcg accagtgtcg cccaggggcg
     2881 actgctggtc gcggcggcga ttagcttcat cgaagacgtc gctcgcacct ggcatctcac
     2941 gcgctacgcc ggagtcgccg acgagatcag tggcctggcg ctggcgatcc tgaccagcgg
     3001 ggcaggtaac ctcttgcgcg catagtaata acattggaag tggataacgg atccgcgatc
     3061 gcggcgcgcc acctggtggc cggccggtac cacgcgtgcg cgctgatccg gctgctaaca
     3121 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     3181 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     3241 atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa
     3301 gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat
     3361 agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg
     3421 gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     3481 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     3541 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     3601 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     3661 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3721 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3781 acaataaccc tgataaatgc ttcaataaca ttgaaaaagg aagagtatga gtattcaaca
     3841 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3901 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3961 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4021 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     4081 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4141 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4201 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4261 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4321 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     4381 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4441 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4501 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4561 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4621 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4681 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4741 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4801 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4861 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4921 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4981 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5041 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5101 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5161 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5221 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5281 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5341 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5401 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5461 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5521 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5581 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     5641 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     5701 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5761 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5821 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5881 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     5941 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     6001 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     6061 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     6121 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     6181 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     6241 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     6301 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     6361 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     6421 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     6481 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
     6541 caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg atggatatgt
     6601 tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg gctccaattc
     6661 ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg
     6721 gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg cgcctacaat
     6781 ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga cgatcagcgg
     6841 tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct gtccctgatg
     6901 gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga
     6961 agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac
     7021 gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     7081 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccg
//
