LOCUS       sequence-205279-        5845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WbpD 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 5845)
  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 5845)
  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-WbpD-His6
FEATURES             Location/Qualifiers
     source          1..5845
                     /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             710..727
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(780..798)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      794..841
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      878..1333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(965..984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1175..1196
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1425..2240)
                     /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(1425..2240)
                     /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     2148..2167
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2232..2251
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2362..2950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2851..2870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3104..3121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3136..3275
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3201..3220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3361..3383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3377..3568)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4377..5459)
                     /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     5421..5440
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5460..5537)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5743..5762
                     /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 atgagttatt atcagcaccc cagcgcgatc gtcgacgacg gtgcgcagat
      181 cggtagcgac tcccgagttt ggcacttcgt gcacatctgt gcaggtgccc ggattggcgc
      241 aggggtttcg ttgggtcaga acgtattcgt cggcaacaag gtcgttattg gtgatcgctg
      301 caagatccag aacaacgtgt cggtatatga caatgtcact ctcgaagagg gcgtgttctg
      361 cgggccgagc atggtattta ccaacgttta caacccccgc tcgttgatcg agcgcaagga
      421 tcagtaccgt aacacgttgg taaaaaaagg tgccacgctt ggtgccaact gcactatcgt
      481 ctgtggcgtg actattggtg aatatgcctt cgtgggtgcg ggtgcggtca ttaacaagaa
      541 tgttccatct tatgccctga tggtaggcgt gcccgctcga cagattggtt ggatgagcga
      601 attcggtgag cagctgcagc tgaacgagca gggcgaagct gtctgctcac actccggtgc
      661 gcgctatgta ctcaatggaa agatcctgag caaggtggac gtgctcgagc accaccacca
      721 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      781 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
      841 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
      901 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      961 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1021 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1081 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1141 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1201 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1261 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1321 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1381 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     1441 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1501 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1561 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1621 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1681 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1741 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1801 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1861 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1921 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1981 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2041 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2101 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2161 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2221 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     2281 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2341 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2401 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2461 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2521 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2581 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2641 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2701 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2761 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2821 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2881 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2941 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3001 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3061 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3121 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3181 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3241 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3301 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3361 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3421 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3481 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3541 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3601 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3661 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3721 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3781 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3841 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3901 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     3961 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4021 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4081 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4141 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4201 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4261 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4321 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4381 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4441 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4501 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4561 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4621 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4681 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4741 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4801 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4861 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4921 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4981 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5041 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5101 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5161 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5221 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5281 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5341 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5401 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5461 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5521 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5581 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5641 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5701 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5761 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5821 gatcgagatc tcgatcccgc gaaat
//
