LOCUS       sequence-389762-        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of C-terminally distrep tag II-mClover3-tagged WzyE from 
            Pectobacterium atrosepticum in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6806)
  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 6806)
  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: pWzyE-strep-mClover3
FEATURES             Location/Qualifiers
     source          1..6806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..130
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(310..1188)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1215..1499
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1422..1441
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2929..2949
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2953..2976
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3013..3036
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             join(3043..3045,3046..3759)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3085..3106)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3693..3714
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3781..3801
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3805..3834
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3878..3895)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4048..4134
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4226..4253
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4272..4363
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4364..4432,4433..5224)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4582..4601)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5266..5721
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5353..5372)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5563..5584
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      5832..6420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6321..6340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6574..6591
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6757..6779
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 aggcagaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
       61 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
      121 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      181 gcgtagagga tctgctcatg tttgacagct tatcatcgat gcataatgtg cctgtcaaat
      241 ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt
      301 cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg
      361 gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc
      421 gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag
      481 cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg
      541 gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat
      601 atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt
      661 atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc
      721 aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat
      781 ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt
      841 attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta
      901 aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc
      961 tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac
     1021 caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc
     1081 gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc
     1141 attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact
     1201 cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt
     1261 cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta
     1321 acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag
     1381 aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc
     1441 cataagatta gcggatctta cctgacgctt tttatcgcaa ctctctactg tttctccata
     1501 cccgtttttt tgggctagca ggaggaatac accatggcgc ttgggcaatt tggagggctg
     1561 tttgtcgtct acctgatttc agtcatcttc atcctgagct tgacctggat ggagtttcgg
     1621 cgcgtacgtt ttaactttaa cgtgctgttc tcgctgctct atttattgac gttctatttt
     1681 ggttttccct ttacctgcgt gctggtgttc cggtttggcg ttgaagtcgt ccccgtgcag
     1741 tatctgcttc aggcgatgtt gtccgcaacg gcgttctatg ctgtctatta cgtcagctac
     1801 aagacgcgac tacgccagaa aacctccgtg ccgcgtgctc cactcctgac ggtgaaccga
     1861 gttgaagcta atctgacctg gctgctgctg gcgttgattg cggtagccac cgtcggtatt
     1921 ttcttcctca acaatggctt cctgctgttt aagctgcgct cttacagcca gatattctct
     1981 agcgatgtgt ccggtgtggc gttgaaacgc tttttctact tcttcattcc ggcaatgctg
     2041 gtggtgtatt tcctgcgcca gacgcagcga gcgtggctac tgttcctcat cggaacggtc
     2101 gcatttggaa tgctgacata tgtgattgtc ggcgggacgc gcgcgaatct gatcattgcc
     2161 ttcgccctat ttctgtttat tggcattgtg cgtggttgga ttacgctctg gatgttggtc
     2221 gctgctggga tatttggtat tgtcgggatg ttctggctgg cgctgaagcg ctatgggctg
     2281 gacgtcagcg gtgattacgc attttatacc ttcctctatt taactcgtga caccttctcg
     2341 ccctgggaga atctggcgct gctgtggcag aattatgaca agattgagtt tcaggggctg
     2401 gcaccgattg ctcgtgattt ctacgtcttt attccctcct ggctatggcc tgaccgtccg
     2461 aatctggtgt taaacagcgc gaactatttc acctgggaag tgttgaataa ccactctggg
     2521 ctggcgatat cccctacgct gttaggctcg ctggtggtga tgggcggcgt gctatttatc
     2581 ccgttgggtg cgattgcggt agggctggtt atcaagtggt tcgactgggt gtacgagtta
     2641 gggaagaacg acagcaatcg ctataaggcc gccattttgc aggcgttctg cttcggtgcg
     2701 gtgttcaata ttattgtgct aacgcgcgaa ggcgtcgatt cctttgtttc acgtgtggtg
     2761 tttttctgtc tgatatttgg cctgtgcctg ctcgttgcca aactgcttta ctggctgctg
     2821 gaaagcgccg gactcatccg gcagcgtctg aggcgcatgc gcgccacgcc gctcgcgccg
     2881 acacccaata ccgtacagga tccggtgata aaggagcaga tactcgagga aaacctgtat
     2941 tttcagggcg cgtggtccca tcctcagttc gagaaaggcg cgggttcagg tgctggcagt
     3001 ggagcgtccg cgtggagtca tccgcaattc gaaaaagaat tcgtgagcaa gggcgaggag
     3061 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3121 ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc
     3181 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     3241 ggcgtggcct gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3301 gccatgcccg aaggctacgt ccaggagcgc accatctctt tcaaggacga cggtacctac
     3361 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3421 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac
     3481 agccactacg tctatatcac ggccgacaag cagaagaact gcatcaaggc taacttcaag
     3541 atccgccaca acgttgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3601 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcca tcagtccaag
     3661 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3721 gccgggatta cacatggcat ggacgagctg tacaagtaga agcttgcggc cgcactcgag
     3781 gaaaacctgt attttcaggg cgcgcaccac caccaccacc atcaccatca ccattgataa
     3841 cgcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac
     3901 gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
     3961 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc
     4021 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     4081 gcctttcgtt ttatctgttg tttgtcagtg aacgctctcc tgagtaggac aaatccgccg
     4141 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     4201 taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt
     4261 ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4321 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4381 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4441 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4501 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4561 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     4621 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4681 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4741 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4801 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4861 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4921 aacattgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4981 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5041 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5101 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5161 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5221 gtaactgtca gaccaagttt actcatatat actttagatt gatttacgcg ccctgtagcg
     5281 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5341 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5401 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5461 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     5521 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5581 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     5641 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5701 aaatattaac gtttacaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5761 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5821 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5881 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5941 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6001 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6061 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6121 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6181 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6241 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6301 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6361 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6421 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6481 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6541 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     6601 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataggg
     6661 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6721 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6781 tttcaccgtc atcaccgaaa cgcgcg
//
