LOCUS       sequence-450327-        5885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express M2 (219-399) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5885)
  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 5885)
  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: pET28b-M2
FEATURES             Location/Qualifiers
     source          1..5885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..74
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(198..245)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     241..259
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(312..329)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(899..931)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(935..952)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(962..979)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             998..1020
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1035..1059
                     /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)."
     primer_bind     complement(1059..1078)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1060..1078)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1162..1181)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1387..1464
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1465..2547
                     /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     complement(1484..1503)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3356..3547
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3541..3563)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3649..3788
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3704..3723
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3803..3820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3974..4562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4054..4073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4673..4692)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4684..5499
                     /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             4684..5499
                     /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     complement(4757..4776)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5591..5885,1..161))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5728..5749)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
       61 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      121 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat
      181 atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
      241 gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca
      301 gccggatctc agtggtggtg gtggtggtgc tcgagtcacc cgctggtcag gatcgccagc
      361 gccaggccac tgatctcgtc ggcgactccg gcgtagcgcg tgagatgcca ggtgcgagcg
      421 acgtcttcga tgaagctaat cgccgccgcg accagcagtc gcccctgggc gacactggtc
      481 gcgggtacca gcttgccgat gaggtcgatc cacacggcct cgcggtcgcc ctgatttcgc
      541 aggtagccgt cgcgtacttc gacagaggcg tgcgacagtt cggtgaccga cactgccacc
      601 agatccggag cgtccaagct gatccgaacg tgcccttgga caaggccgcg caaccgttgt
      661 gccgcttgct gattcgctcg tagcgctcgg atgcactcca ggcagcgcca ctcgtcgagg
      721 cggcggatga gcgcgtccag gatggcctgt ttggaagaaa acgaacggta cagccccggg
      781 cccgcgatgc cggctccctt gccgatttcg ctggtgttga cggccggata gccctgcgca
      841 cggaacagcc gcgcgcccgc ggccagcagg gtctcgtagc gcatgaattc ggatcccgac
      901 ccatttgctg tccaccagtc atgctagcca tatggctgcc gcgcggcacc aggccgctgc
      961 tgtgatgatg atgatgatgg ctgctgccca tggtatatct ccttcttaaa gttaaacaaa
     1021 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     1081 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     1141 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     1201 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     1261 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
     1321 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     1381 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     1441 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     1501 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     1561 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     1621 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     1681 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     1741 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     1801 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     1861 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     1921 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     1981 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     2041 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     2101 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     2161 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     2221 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     2281 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     2341 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     2401 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     2461 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2521 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     2581 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     2641 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     2701 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     2761 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     2821 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     2881 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     2941 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     3001 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     3061 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     3121 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     3181 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     3241 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     3301 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     3361 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     3421 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     3481 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3541 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3601 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3661 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     3721 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     3781 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3841 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3901 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3961 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4021 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4081 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4141 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4201 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4261 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4321 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4381 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4441 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4501 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4561 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4621 aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     4681 gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     4741 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     4801 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     4861 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     4921 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     4981 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     5041 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     5101 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     5161 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     5221 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     5281 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     5341 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     5401 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     5461 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     5521 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5581 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5641 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     5701 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5761 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5821 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5881 aaagg
//
