LOCUS       sequence-450318-        5885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express M1 (13-193) 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-M1
FEATURES             Location/Qualifiers
     source          1..5885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(442..1524)
                     /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     1486..1505
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1525..1602)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1808..1827
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1911..1930
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1911..1929
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1930..1954
                     /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             1969..1991
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2010..2027
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2037..2054
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2058..2090
                     /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             2660..2677
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2730..2748)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2744..2791
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2828..3283
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2915..2934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3125..3146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3375..4190)
                     /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             complement(3375..4190)
                     /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     4098..4117
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4182..4201
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4312..4900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4801..4820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5054..5071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5086..5225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5151..5170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5311..5333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5327..5518)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
       61 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
      121 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
      181 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
      241 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
      301 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
      361 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
      421 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      481 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
      541 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
      601 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
      661 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
      721 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
      781 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
      841 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
      901 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
      961 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     1021 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     1081 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     1141 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     1201 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     1261 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     1321 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     1381 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     1441 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     1501 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     1561 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     1621 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     1681 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     1741 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     1801 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     1861 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     1921 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     1981 taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcctgg
     2041 tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt cgggatccga
     2101 attcatgaag gaccggaaga agcagatttt ggatcaggcc gttggactgt tcatcgaacg
     2161 tggcttccat tcggtcaaat tggaggacat tgccgaggcg gccggggtga ccgcgcgcgc
     2221 gttgtatcgc cactacgaca acaagcaggc gttgctcgcc gaagcgatcc gaaccggcca
     2281 ggatcagtac cagagcgcgc gtcgtctcac cgagggcgag acggagccga cgccgcggcc
     2341 gttgaacgcc gatctggaag acctgatcgc cgcggcggtc gcctctcggg cgttgacggt
     2401 gctgtggcag cgcgaggccc gctacctcaa cgaggacgac cgcacggcgg tccggcgccg
     2461 catcaacgcg atcgtcgccg gcatgcgtga cagcgtgctg ctggaggtgc ccgatctgag
     2521 tccacagcat tcggagttgc gggcgtgggc ggtgtccagc actttgacca gcctgggccg
     2581 gcacagccta agcctgccgg gcgaggaact gaaaaagctt ctctaccagg cgtgtatggc
     2641 cgcggcaagg tgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     2701 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     2761 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     2821 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2881 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2941 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3001 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3061 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3121 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3181 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3241 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     3301 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3361 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     3421 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     3481 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     3541 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     3601 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     3661 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     3721 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     3781 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     3841 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     3901 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     3961 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     4021 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     4081 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     4141 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     4201 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     4261 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4321 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4381 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4441 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4501 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4561 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4621 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4681 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4741 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4801 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4861 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4921 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4981 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5041 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     5101 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     5161 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     5221 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5281 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5341 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     5401 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     5461 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     5521 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     5581 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     5641 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     5701 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     5761 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     5821 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     5881 ttcac
//
