LOCUS       sequence-450316-        6366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express MelJ (Rv1940) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6366)
  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 6366)
  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-MelJ
FEATURES             Location/Qualifiers
     source          1..6366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(145..1227)
                     /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     1189..1208
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1228..1305)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1511..1530
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1614..1633
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1614..1632
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1633..1657
                     /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             1672..1694
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1713..1730
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1740..1757
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2844..2861
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2914..2932)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2928..2975
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3012..3467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3099..3118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3309..3330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3559..4374)
                     /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(3559..4374)
                     /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     4282..4301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4366..4385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4496..5084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4985..5004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5238..5255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5270..5409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5335..5354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5495..5517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5511..5702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
       61 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
      121 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
      181 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
      241 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
      301 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
      361 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
      421 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
      481 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
      541 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
      601 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
      661 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
      721 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
      781 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
      841 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
      901 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
      961 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     1021 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     1081 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1141 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     1201 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     1261 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     1321 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     1381 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     1441 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     1501 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     1561 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     1621 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     1681 ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc
     1741 tggtgccgcg cggcagccat atgaagacga ccgatgtgcg ggtacgtcgt gcgatcacgg
     1801 cgatggcggg cggtcacgcc gtggtcctga ccggcgaccc caatggcgat ggctatctcg
     1861 tcttcgccgc ccaggccgcg acgccgcggc tggttgcctt tgcggtccgg cacacctcgg
     1921 gttatttgcg cgtcgcgctg ccgggcgccg aatgcgagcg actgcacctg ccgcccatgt
     1981 gtgaccgaga caccacgcat tgcgtgtcgg tcgacgttcg cggcaccggc accggaatct
     2041 cggcgagcga tcgcgcctgg accatcgcgg cactggcttc ggccacctcc gtcgccgccg
     2101 atttccaacg tccgggccat gtggtgcccg tgcaggcgca agccgacggt gtgctgggtc
     2161 ggcggggacc cgccgaggcg gccgtcgacc tggcccgcct ggcggaacgg cggccggccg
     2221 ccgcgctctg cgagatcgtc tcgcccgata atcccgtcca gatggcgcac cacgccgagt
     2281 cggtcgaatt cgccgtcgaa cacggactgg ccatggtctc gatcggggag ctggtggcgt
     2341 atcgccggcg gatcgagccc caggtggtcc ggtttacggc agcgacgctg cccacctggg
     2401 ccggcgcctc gcgtgtcatc ggctttcgtg acgtttacga cctcggcgag catttggcgg
     2461 tcatcgtggg tgcggtcggt gccggggtgc ccgtgccgct gcacgtccac atcgagtgcc
     2521 tgacgggcga cgtgttcggc tcgacggcgt gccgctgcgg cgaggaactc aacggcgcgc
     2581 tggcgaggat gtcggctcag ggcagcggcg tggtcttgta tctgcgtccg cccggacccg
     2641 cgcaagcgtg cggcttgttc gcccggggcg atgcggcgac cgatgtcatg ccggagaccg
     2701 tgacatggat cctgcgcgat cttggggtgt atgcgatccg actttccgat gatgtgccag
     2761 gatttgggct tgtcatgttc ggggcgatcc gagaagccag cacgttggcg gccgcaggtt
     2821 gaaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     2881 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2941 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     3001 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3061 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     3601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     3661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     3721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     3781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     3841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     3901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     3961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     4381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     4441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     5341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     5881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     5941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     6001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     6061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     6121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     6181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     6241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     6301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     6361 tgcatg
//
