LOCUS       sequence-243951-        6295 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Overexpression plasmid for production of WcbI from Burkholderia 
            pseudomallei.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6295)
  AUTHORS   Vivoli M, Ayres E, Beaumont E, Isupov MN, Harmer NJ
  TITLE     Structural insights into WcbI, a novel polysaccharide-biosynthesis 
            enzyme.
  JOURNAL   IUCrJ. 2013 Oct 18;1(Pt 1):28-38. doi: 10.1107/S205225251302695X. 
            eCollection 2014 Jan 1.
  PUBMED    25075317
REFERENCE   2  (bases 1 to 6295)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "IUCrJ."; 
            date: "2013-10-18"; volume: "1(Pt 1):28-38. doi"; pages: " 
            10.1107/S205225251302695X. eCollection 2014 Jan 1"
COMMENT     Sequence Label: pNIC28-BpWcbI
FEATURES             Location/Qualifiers
     source          1..6295
                     /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             1160..1177
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1230..1248)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1244..1291
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1328..1783
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1415..1434)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1625..1646
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1875..2690)
                     /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(1875..2690)
                     /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     2598..2617
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2682..2701
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2812..3400
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3301..3320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3554..3571
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3586..3725
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3651..3670)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3811..3833
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3827..4018)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4827..5909)
                     /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     5871..5890
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5910..5987)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6193..6212
                     /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 atccatggca caacgaaaga aatattcggt
      181 gtacggctcg tgtcaggcgc cggcgttagc caagatgctg aacagttgtc cgacattcgc
      241 ccgcgactgg gaactggtgg agatggagcc gtgctttgtc gccagcgaag agcaaatcga
      301 tcgtcatctc gcggaaacga taccgaagct cgatctattt ctttatcaac ctgtttcgga
      361 ggggtatcga ggagaaaagt actcgtcggt atttctgagg aatagcatgc cgccgggagg
      421 gaacgccttg tcggtgcaat acatgcattg ggaaggctat catcccaccg tcaattcacc
      481 atacggtttg ccgccgcatc cggaaggtta tgttgatgcc ttgatcgcgg gagccgtcgt
      541 catggatgtg gacaaggaaa cctatctccg gcacttggag gagatcggcg cgtctctacg
      601 catcgatatc gacgagatcg aaagctggtg cgtcgacgag ctgaagactc gggaagtcgg
      661 tgaaaacgac ggtgggaagc agatcgacat ttccgttacc gattttattt tggctaactg
      721 tcgtcagaag cgactgttct acacgatgaa tcatccgacc gcggcattga tgcgcgaaat
      781 cgcggcacga tgcatgctgg cgctcggcta tacctattcg gatatcagct ttgatcagaa
      841 tctcgatcct ctggatgtga ccaagatgtc cttgtatccg atatatcgag attgcttcga
      901 tttttcagaa ttgaacagga tgaacgagta tcaggtgctt tacaagaaga aggcctacga
      961 accgtatctc ctggagcagt tcgagtggtt tgagcgctcg ccgaaggccg acgtttccgc
     1021 gttcttcgat cgtgtcgccg caaatcgtcg ctgggttcga acggcgttgc gcagggcttt
     1081 tgaatcatga taagcttcag taaaggtgga tacggatccg aattcgagct ccgtcgacaa
     1141 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     1201 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1261 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1321 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1381 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1441 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1501 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1561 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1621 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1681 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1741 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1801 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1861 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     1921 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     1981 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     2041 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     2101 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     2161 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     2221 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     2281 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2341 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     2401 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2461 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2521 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     2581 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     2641 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     2701 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     2761 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2821 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2881 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2941 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3001 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3061 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3121 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3181 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3241 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3301 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3361 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3421 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3481 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3541 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3601 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3661 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3721 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3781 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3841 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3901 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3961 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     4021 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4081 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4141 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4201 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4261 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4321 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4381 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4441 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4501 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4561 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4621 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4681 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4741 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4801 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4861 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4921 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4981 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     5041 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     5101 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5161 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5221 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5281 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5341 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5401 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5461 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5521 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5581 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5641 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5701 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5761 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5821 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5881 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5941 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     6001 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     6061 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6121 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     6181 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     6241 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaat
//
