LOCUS       sequence-205285-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WbpB from P. aeruginosa with an N-terminal T7 tag and a 
            C-terminal His6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  AUTHORS   Zamora CY, Schocker NS, Chang MM, Imperiali B
  TITLE     Chemoenzymatic Synthesis and Applications of Prokaryote-Specific 
            UDP-Sugars.
  JOURNAL   Methods Enzymol. 2017;597:145-186. doi: 10.1016/bs.mie.2017.06.003. 
            Epub 2017 Jul 5.
  PUBMED    28935101
REFERENCE   2  (bases 1 to 6220)
  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"; journalName: "Methods 
            Enzymol."; date: "2017"; volume: "597:145-186. doi"; pages: " 
            10.1016/bs.mie.2017.06.003. Epub 2017 Jul 5"
COMMENT     Sequence Label: T7-WbpB-His6
FEATURES             Location/Qualifiers
     source          1..6220
                     /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             89..121
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1085..1102
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1155..1173)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1169..1216
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1253..1708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1340..1359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1550..1571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1800..2615)
                     /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(1800..2615)
                     /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     2523..2542
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2607..2626
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2737..3325
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3226..3245
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3479..3496
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3511..3650
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3576..3595)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3736..3758
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3752..3943)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4752..5834)
                     /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     5796..5815
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5835..5912)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6118..6137
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggctagcatg actggtggac agcaaatggg
      121 tcgcggatcc atgaaaaatt tcgctctcat cggtgctgcc ggctacatcg ctcctcgcca
      181 tatgcgcgcc atcaaagaca ccggtaactg cctggtttcg gcctatgaca tcaatgactc
      241 ggtcggtatt attgatagca tctctcccca gagcgagttt tttaccgagt tcgagttctt
      301 tcttgatcat gcgagcaacc tcaagcgcga ctctgctacc gcgctggact acgtatcgat
      361 ctgctcgccc aattacctgc actacccgca tatcgctgca ggtctgcgct tgggttgcga
      421 cgtaatctgc gaaaagccgc ttgttccaac cccagagatg ctcgatcagt tggctgttat
      481 cgagcgcgaa accgataagc gcctctacaa cattctgcaa ctgcgtcatc accaggcgat
      541 catcgcattg aaggacaagg tcgcccgcga aaaaagtccg cataagtacg aggtcgatct
      601 gacttacatt acttcccgcg gcaactggta tctgaaaagc tggaagggag atccacgtaa
      661 gtcgttcggc gtggctacca acatcggtgt gcacttctac gacatgctgc acttcatctt
      721 tggcaagctg cagcgtaatg ttgtgcactt cacttccgag tacaaggcag ctggttatct
      781 ggagtacgag caggcccgtg tgcgttggtt tctgtccgtg gatgctaacg acctgccgga
      841 gtcggtcaag ggcaaaaagc cgacctatcg ttcgattacc gtcaacggtg aggaaatgga
      901 gttctctgaa ggctttaccg atctacatac aaccagctac gaagaaattc tcgctggtcg
      961 tggttatggc atcgatgacg ctcgtcattg tgtggaaact gtcaatacca ttcgcagcgc
     1021 cgtcatcgta ccggcctctg ataacgaagg gcatccgttc gtcgcggcgc ttgcgcgtct
     1081 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1141 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1201 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1261 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1321 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1381 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1441 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1501 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1561 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1621 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1681 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     1741 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     1801 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     1861 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     1921 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     1981 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2041 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     2101 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2161 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2221 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2281 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2341 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2401 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2461 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2521 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2581 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2641 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2701 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2761 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2821 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2881 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2941 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3001 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3061 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3121 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3181 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3241 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3301 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3361 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3421 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3481 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3541 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3601 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3661 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3721 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3781 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3841 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3901 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     3961 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4021 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4081 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4141 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4201 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4261 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4321 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4381 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4441 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4501 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4561 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4621 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4681 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4741 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4801 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4861 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     4921 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     4981 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5041 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5101 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5161 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5221 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5281 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5341 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5401 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5461 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5521 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5581 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5641 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5701 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5761 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5821 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     5881 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     5941 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6001 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6061 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6121 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6181 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
