LOCUS       sequence-450326-        7824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express MelG (Rv1937) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7824)
  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 7824)
  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-MelG
FEATURES             Location/Qualifiers
     source          1..7824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(410..1492)
                     /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     1454..1473
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1493..1570)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1776..1795
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1879..1898
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1879..1897
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1898..1922
                     /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             1937..1959
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1978..1995
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2005..2022
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4567..4584
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4637..4655)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4651..4698
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4735..5190
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4822..4841)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5032..5053
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5282..6097)
                     /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(5282..6097)
                     /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     6005..6024
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6089..6108
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6219..6807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6708..6727
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6961..6978
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6993..7132
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7058..7077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7218..7240
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7234..7425)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
       61 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
      121 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
      181 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
      241 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
      301 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
      361 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
      421 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      481 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
      541 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
      601 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
      661 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
      721 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
      781 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
      841 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
      901 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
      961 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1021 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1081 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1141 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1201 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1261 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     1321 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     1381 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     1441 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1501 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     1561 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     1621 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     1681 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     1741 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     1801 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     1861 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1921 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
     1981 catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc ggttcgtcag
     2041 gtcaccgtcg gctattcgga cggcacgcac aagacgatgc cggtgcggtg cgaccagacg
     2101 gtcctggatg ccgccgagga acacggcgtg gccatcgtca acgaatgcca aagcgggata
     2161 tgtggcacct gcgtggccac ctgcaccgcc ggccgctacc agatgggacg caccgaggga
     2221 ctgtccgatg tcgagcgggc ggcgcgaaag atcctcacct gccagacgtt tgttacctcc
     2281 gattgccgga tcgagctgca gtatccggtc gacgacaacg ccgccctgct ggtcaccggt
     2341 gacggtgtgg tgaccgcggt cgagttggtg tcgcccagca ccgccatcct gcgggtggac
     2401 acctctggca tggccggcgc gctgagatac cgggccggcc agttcgccca attgcaggtt
     2461 cccggtacca acgtatggcg caactactcc tacgcccatc cggccgacgg ccgcggtgag
     2521 tgcgagttca tcatcaggtt gctgccggac ggcgtgatgt cgaattatct tcgcgaccgc
     2581 gcccagcccg gtgaccatat cgcgctgcgc tgcagcaagg gcagctttta tctgcgcccg
     2641 atcgtgcgac cggtgatcct ggtcgccgga ggaaccggcc tgtcagcgat cctggcgatg
     2701 gcccagagcc tggatgccga tgtcgctcac ccggtctacc tgctctacgg ggtcgagcgc
     2761 accgaagacc tgtgcaagct cgacgaactc accgagctgc gccgccgcgt tggccgcctg
     2821 gaggtgcacg tcgtcgtcgc tcgcccggac cccgactggg atgggcgcac cgggctggtc
     2881 accgacctgc tcgacgagcg gatgctggcg agcggtgacg ccgacgtgta tctgtgcggt
     2941 ccggtcgcca tggtcgacgc agcccgaacc tggctggacc acaatggctt tcaccgtgtc
     3001 gggttgtact acgagaagtt cgtggccagc ggggcggcgc gccgccgcac cccggctcgg
     3061 ctggattacg cgggcgtgga cattgccgag gtgtgccgcc gcggccgcgg caccgcggtg
     3121 gtcatcggcg gcagcatcgc gggcatcgcg gcggcgaaaa tgctcagcga gaccttcgat
     3181 cgcgtcatcg tgctggagaa ggacggcccg caccgtcgcc gcgagggcag gccgggcgcg
     3241 gcacagggtt ggcacctgca ccacctgctg accgccgggc agatcgagct ggagcgcatc
     3301 ttccctggca tcgtcgacga catggtgcgc gagggagcgt tcaaggtcga catggccgcg
     3361 cagtaccgta tccggctggg cggcacctgg aagaagcccg gcactagtga catcgagatc
     3421 gtctgcgcgg gaaggccgct gctcgaatgg tgtgtgcgcc gccggctcga cgacgaaccg
     3481 cgcatcgact tccgctacga atcggaggtg gccgatctcg ccttcgaccg cgccaacaat
     3541 gccatcgtcg gcgtcgccgt ggacaatggc gacgccgacg gaggcgacgg tttgcaggtg
     3601 gtgcccgccg agttcgtcgt ggacgcgtcg ggcaagaaca cccgcgtgcc ggagttcttg
     3661 gagcgtctcg gtgttggcgc tcccgaggcc gagcaggaca tcatcaactg cttctactcc
     3721 acgatgcagc accgggttcc gccggagcgg cggtggcagg acaaggtgat ggtgatctgc
     3781 tatgcgtacc gccctttcga ggatacctac gccgcgcagt actacaccga cagctcccgc
     3841 accatcctgt ccacctcact ggtggcctac aactgctatt cgccgccgcg taccgcccga
     3901 gaattccgcg cgttcgccga cctgatgccg tccccggtca tcggggagaa catcgacggg
     3961 ctggagccgg catcgcccat ctacaatttc cgctatccca acatgctgcg gctgcgctac
     4021 gagaagaagc gcaacctgcc gcgggctttg ctggcggtgg gcgatgccta caccagcgcc
     4081 gacccggtgt cgggtctggg tatgagcctg gcgctcaagg aagttcggga gatgcaggcg
     4141 ctgctggcta aatacggcgc cggtcaccgg gatctgccgc gccggtacta ccgggcgatc
     4201 gccaagatgg ccgacacggc ctggttcgtg atccgcgagc agaacctgcg cttcgactgg
     4261 atgaaggacg tcgacaagaa gcgcccgttc tatttcggtg tgctgacctg gtacatggac
     4321 cgcgtgctgg agctggtgca tgacgatctc gacgcgtacc gggaattctt ggccgtcgtc
     4381 catctggtca agccgccgtc ggcgctgatg cgacccagga tcgccagccg cgtcctcggc
     4441 aaatgggcac gaacccgatt gtcgggccag aagacgttga ttgcccgcaa ctacgaaaat
     4501 catccgatac cagccgaacc cgcggaccaa cttgtaaacg cttagaagct tgcggccgca
     4561 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     4621 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     4681 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     4741 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4801 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4861 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4921 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4981 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5041 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5101 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5161 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     5221 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     5281 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     5341 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     5401 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     5461 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     5521 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     5581 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     5641 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     5701 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     5761 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     5821 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     5881 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     5941 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     6001 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     6061 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     6121 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6181 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6241 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6301 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6361 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6421 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6481 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6541 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6601 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6661 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6721 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6781 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6841 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6901 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6961 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     7021 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     7081 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     7141 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     7201 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     7261 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     7321 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     7381 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     7441 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     7501 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     7561 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     7621 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     7681 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     7741 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     7801 gcgtatcggt gattcattct gcta
//
