LOCUS       sequence-244090-        6400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression plasmid for production of WcbL from Burkholderia 
            pseudomallei.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6400)
  AUTHORS   Vivoli M, Isupov MN, Nicholas R, Hill A, Scott AE, Kosma P, Prior 
            JL, Harmer NJ
  TITLE     Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to 
            Drug Discovery.
  JOURNAL   Chem Biol. 2015 Dec 17;22(12):1622-32. doi: 
            10.1016/j.chembiol.2015.10.015.
  PUBMED    26687481
REFERENCE   2  (bases 1 to 6400)
  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: 
            "10.1016/j.chembiol.2015.10"; journalName: "Chem Biol"; date: 
            "2015-12-17- 17"; volume: "22"; issue: "12"; pages: "1622-32"
COMMENT     Sequence Label: pNIC28-BpWcbL
FEATURES             Location/Qualifiers
     source          1..6400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1265..1282
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1335..1353)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1349..1396
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1433..1888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1520..1539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1730..1751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1980..2795)
                     /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(1980..2795)
                     /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     2703..2722
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2787..2806
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2917..3505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3406..3425
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3659..3676
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3691..3830
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3756..3775)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3916..3938
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3932..4123)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4932..6014)
                     /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     5976..5995
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6015..6092)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6298..6317
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgaat ccgacaatca ttcgcgcacg
      181 ggccccgctg cggttgggcc tggcgggagg ggggaccgac gtcgctccct atgccgatac
      241 attcgggggt tatgtcctga atgcgacgat tgatcgctac gcatacgcgg tgatcaagac
      301 gctcacgata ccggccgtgc ggttcgtctc gacggatcag caggtggaaa agcatcagct
      361 catttccgag ccgctggagc tgaacggtac gctgaacctg cataaggcgg tgtacaacca
      421 catgattcgg aactacaacc atggcaagcc gatcgcgctc gagctgagta cattctgtga
      481 cgctcccgcg ggttccgggt tgggttcgtc gtcgacgctg gtcgtcgtga tgatcaaggc
      541 gtttgtcgag ttgctgaatc tgccgctgga cgactacgcc atcgcgcagc tggcttatcg
      601 gatcgagcgc gtcgattgcg ggctggccgg gggacgtcag gaccaatatt cggcgacgtt
      661 cgggggcttc aatttcatgg agttctatga ggaagaacgg acgatcgtga atccgttgcg
      721 aatcaagaac tgggttcttt gtgagctcga ggcctcgctg gtcctttttt acacgggcgt
      781 gtcgcgcgag tcggccaaga tcattcagga ccagagcgac aacgtggtgt cgcacaagac
      841 cgcggcaatc gaggcgatgc acggtatcaa gcgcgaggcg ctggtcatga aagaggcttt
      901 gctgaagggc gatttcaagg cctttgtcgc ttcgatgcgc cttggttggg acaacaagaa
      961 gaattcggcg cgaactgttt ccaacgcgca tatcgacgag atctacgacg cggcgatccg
     1021 cgccggtgcg caggccggaa aggtttcggg tgcgggcggc ggcggtttca tgctgttttt
     1081 cgtacccacc gagaagcgca tggatctgat caggacgctt ggcgaatacg acggacaagt
     1141 tagcaactgc cactttacga agaacgggac gcaggcatgg agaatcgcga attgataagc
     1201 ttcagtaaag gtggatacgg atccgaattc gagctccgtc gacaagcttg cggccgcact
     1261 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1321 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1381 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1621 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1741 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1861 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     1921 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     1981 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2041 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2101 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2161 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2221 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     2281 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2341 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2401 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2461 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2521 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2581 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2641 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2701 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2761 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2821 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2881 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2941 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3001 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3061 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3121 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3181 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3241 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3301 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3361 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3421 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3481 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3541 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3601 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3661 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3721 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3781 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3841 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3901 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3961 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4021 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4081 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4141 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4201 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4261 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4321 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4381 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4441 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4501 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4561 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4621 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4681 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4741 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4801 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4861 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4921 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4981 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5041 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5101 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5161 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5221 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5281 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5341 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5401 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5461 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5521 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5581 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5641 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5701 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5761 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5821 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5881 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5941 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6001 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6061 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6121 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6181 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6241 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6301 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6361 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
