LOCUS       sequence-450319-        6078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express MelK (Rv1941) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6078)
  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 6078)
  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-MelK
FEATURES             Location/Qualifiers
     source          1..6078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     159..176
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    191..330
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(256..275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     416..438
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(432..623)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1432..2514)
                     /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     2476..2495
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2515..2592)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2798..2817
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2901..2920
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2901..2919
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2920..2944
                     /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             2959..2981
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3000..3017
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3027..3044
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3843..3860
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3913..3931)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3927..3974
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4011..4466
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4098..4117)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4308..4329
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4558..5373)
                     /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(4558..5373)
                     /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     5281..5300
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5365..5384
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5495..6078,1..5)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5984..6003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
       61 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      121 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      181 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      241 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      301 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
      361 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
      421 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
      481 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
      541 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
      601 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
      661 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
      721 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
      781 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
      841 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
      901 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
      961 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1021 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1081 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1141 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1201 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1261 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1321 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1381 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1441 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1501 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1561 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     1621 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     1681 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     1741 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     1801 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     1861 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     1921 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     1981 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2041 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2101 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2161 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2221 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2281 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2341 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2401 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2461 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2521 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     2581 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     2641 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     2701 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     2761 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     2821 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     2881 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     2941 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
     3001 atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg ttgaaccatc
     3061 cagacctggc cggcaaggtc gcgatcgtta ctggggcggg cgccggaatc ggtctggcgg
     3121 ttgcccggcg actcgccgac gagggctgcc atgtgctgtg cgcggacatc gatggtgatg
     3181 ccgcggatgc cgcggccacc aaaatcggtt gtggcgcagc ggcctgccgg gttgacgtca
     3241 gcgacgaaca acagatcatc gccatggtcg acgcctgtgt tgccgcgttc ggcggggtgg
     3301 acaagttggt cgccaacgcc ggtgtcgttc atctggcttc gctcatcgac accaccgtcg
     3361 aggacttcga tcgggtcatc gcgatcaatc tccgcggcgc ctggctgtgc accaagcatg
     3421 cggcaccgcg gatgatcgag cgcggcgggg gagccattgt caacctgtcg tcgttagcgg
     3481 gccaggtagc ggtgggcggc accggcgcat acggcatgtc gaaggccggc atcatccagc
     3541 tcagccgcat caccgccgcc gaactgcgct cgtcgggcat ccgctccaac acgctgctgc
     3601 ccgcattcgt cgacaccccg atgcagcaga ccgccatggc aatgttcgac ggggccctgg
     3661 gcgcgggggg tgcgcgctcg atgattgccc ggctgcaggg ccgcatggcc gcaccggagg
     3721 agatggccgg catcgtggtg ttcctgctgt ccgacgatgc gtcgatgatc accggcacca
     3781 cccagatcgc cgacggcggg acgattgccg cgctgtggtg aaagcttgcg gccgcactcg
     3841 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     3901 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3961 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     4021 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4081 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4141 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4201 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4261 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4321 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4381 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4441 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     4501 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     4561 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     4621 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     4681 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     4741 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     4801 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     4861 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4921 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4981 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     5041 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     5101 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     5161 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     5221 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     5281 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     5341 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     5401 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5461 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5521 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5581 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5641 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5701 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5761 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5821 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5881 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5941 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6001 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6061 caggggggcg gagcctat
//
