LOCUS       sequence-389743-        6360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N-terminally His6-tagged WzzE from Pectobacterium 
            atrosepticum in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6360)
  AUTHORS   Weckener M, Woodward LS, Clarke BR, Liu H, Ward PN, Le Bas A, Bhella
            D, Whitfield C, Naismith JH
  TITLE     The lipid linked oligosaccharide polymerase Wzy and its regulating 
            co-polymerase, Wzz, from enterobacterial common antigen biosynthesis
            form a complex.
  JOURNAL   Open Biol. 2023 Mar;13(3):220373. doi: 10.1098/rsob.220373. Epub 
            2023 Mar 22.
  PUBMED    36944376
REFERENCE   2  (bases 1 to 6360)
  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: "Open 
            Biol."; date: "2023-03"; volume: "13(3):220373. doi"; pages: " 
            10.1098/rsob.220373. Epub 2023 Mar 22"
COMMENT     Sequence Label: pHis-WzzE
FEATURES             Location/Qualifiers
     source          1..6360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1053..1072
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1092..1169)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1375..1394
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1482..1501
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1482..1500
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1501..1525
                     /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             1540..1562
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1582..1599
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1621..1641
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2701..2718
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2771..2789)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2785..2832
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2869..3324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2956..2975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3166..3187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3416..4231)
                     /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(3416..4231)
                     /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     4139..4158
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4223..4242
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4353..4941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4842..4861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5095..5112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5127..5266
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5192..5211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5352..5374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5368..5559)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
       61 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggnt ttttcttttc
      121 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
      181 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
      241 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
      301 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
      361 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
      421 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
      481 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
      541 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
      601 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
      661 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
      721 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
      781 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
      841 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
      901 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
      961 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     1021 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     1081 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     1141 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     1201 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     1261 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     1321 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     1381 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     1441 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg
     1501 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     1561 gatatacata tgtcgtacta ccatcaccat caccatcacg attacgatat cccaacgacc
     1621 gaaaacctgt attttcaggg cgccatggtg aaatcagaga acttgtctac cgggaatgcg
     1681 ttgattgata acgaactgga tatccgtggt ttattccgcc tgctgtggcg tggaaaggtg
     1741 tggccgatag caatcggcct gctgtttgcc gtcgtggcat taggctattc ctatcttgtt
     1801 aaacaggaat ggggtgcaac atcgattacc gacaagccga cggttaatat gttgggcggc
     1861 tattactcgc agcagcaatt cctacgtaac ctcgacgctc gatcgttttc ctctcctcag
     1921 ccagaacaac cgtctatttc ggctggtgct tatgatgaat tcatcatgca gttagcggct
     1981 tacgataccc gccgcgattt ctggttgcag actgactatt ataagcaacg tctggaaggt
     2041 gatgaaaagg ctgatgcagc cttgctggat gagctggtca acaacattca gtttacggcg
     2101 cgtgatgacg ggaaaaaaac caatgactcc gtgaagctgg tggcggaaac ctcctcagat
     2161 tccaacacgt tgcttcgtca gtatgtggtt tttgccagtc agcgtgcagc caaccacttg
     2221 aatgaagaaa tcaagggagc ctgggctgcc agaaccattt tcatgaagtc gcagattaag
     2281 cgtcaggaag cggtagcaaa agcgatttac gatcgtgaag tccgcagcgt tgaactggcg
     2341 ctgaaaatcg cacaacagca aggtattagc cgtagccaga cggatacgcc agcagatgaa
     2401 atccccgcat cggaaatgtt cctgctcggc aggccgatgc ttcaagcccg actggaaacg
     2461 ttgcagacca gcggcccgca ttatgaactg gattacgatc aaaatcgcgc gatgttggcg
     2521 acgctgaacg ttggcccaac gcttgaggcc agtttccaga cgtatcgtta tttgcgtaca
     2581 ccggaagaac cggtgaaacg cgacagcccg cgccgggcat tcctgatggt gatgtggggt
     2641 gcgattggtg tgctagtggg agcgggtgtt gccctggctc gccgtctgcg ttagctcgag
     2701 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     2761 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2821 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2881 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2941 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3001 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3061 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3121 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3181 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3301 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3361 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     3421 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     3481 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     3541 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     3601 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     3661 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     3721 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     3781 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     3841 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     3901 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     3961 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     4021 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     4081 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     4141 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     4201 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     4261 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4321 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4381 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4441 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4501 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4561 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4621 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4681 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4741 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4801 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4861 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4921 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4981 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5041 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5101 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5161 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5221 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5281 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5341 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5401 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5461 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5521 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5581 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5641 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5701 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5761 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5821 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5881 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     5941 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     6001 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     6061 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     6121 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     6181 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     6241 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     6301 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
//
