LOCUS       sequence-450320-        5823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express MelI (Rv1939) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5823)
  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 5823)
  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-MelI
FEATURES             Location/Qualifiers
     source          1..5823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(230..1045)
                     /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(230..1045)
                     /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     953..972
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1037..1056
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1167..1755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1656..1675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1909..1926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1941..2080
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2006..2025)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2166..2188
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2182..2373)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3182..4264)
                     /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     4226..4245
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4265..4342)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4548..4567
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4651..4670
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4651..4669
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4670..4694
                     /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             4709..4731
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4750..4767
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4777..4794
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5338..5355
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5408..5426)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5422..5469
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5506..5823,1..138)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5593..5612)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(5803..5823,1)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
       61 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      121 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      181 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
      241 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
      301 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
      361 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
      421 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
      481 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
      541 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
      601 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
      661 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
      721 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
      781 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
      841 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
      901 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
      961 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     1021 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     1081 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1141 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1201 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1261 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     1321 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1381 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1441 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1501 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1561 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1621 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1681 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1741 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1801 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     1861 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     1921 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     1981 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     2041 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     2101 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2161 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     2221 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     2281 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     2341 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     2401 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     2461 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     2521 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     2581 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     2641 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     2701 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     2761 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     2821 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     2881 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     2941 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     3001 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     3061 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     3121 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     3181 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3241 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     3301 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     3361 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     3421 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     3481 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     3541 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     3601 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     3661 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     3721 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     3781 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     3841 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     3901 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     3961 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4021 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     4081 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     4141 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     4201 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     4261 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     4321 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     4381 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     4441 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     4501 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     4561 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     4621 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     4681 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
     4741 ggcagcagcc atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg
     4801 gtgagctgca ccttcgacat ggtcccggag accgtcgatc atctcgacga ggtcgggctg
     4861 cggcgggtct tcggctgctt tccgtgcggc gtgatcgccg tctgcgcgat ggtcgacgac
     4921 cagccggtcg gcatggcggc cagctcgttc acgtcggttt cagttgaccc gccgctggta
     4981 tcgatctgtg tgcagaactg ttcgacgacg tggccgaagt tgcgcgaccg cccacggctc
     5041 ggtgtgagcg tgctcgccga ggggcacgac gcggcctgta tgagcctgtc gcgcaaggaa
     5101 ggtaaccggt tcgccggggt gttctggagc gaattgtcca gcgggggtgt ggtgatcgcc
     5161 ggggccggcg cctggctgga ttgccgcccg tacgcggaga tcccggcggg ggatcacctg
     5221 atcgccctgc tggagatctg cgcggtgcgc gccgatcccg agacaccgcc gctggtgttt
     5281 cacggtagcc ggttccgccg gttggagtct cgatgaaagc ttgcggccgc actcgagcac
     5341 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     5401 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     5461 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     5521 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5581 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5641 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5701 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     5761 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5821 ctg
//
