LOCUS       sequence-389748-        5518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N-terminally His6-mCherry-tagged WzzE from 
            Pectobacterium atrosepticum in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5518)
  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 5518)
  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-mCherry-WzzE
FEATURES             Location/Qualifiers
     source          1..5518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..131)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(248..793)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(328..347)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1018..2100)
                     /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     2062..2081
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2101..2178)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2224..2243
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2224..2242
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2243..2267
                     /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)."
     CDS             2323..2340
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2495..2513)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2763..2782
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4114..4158
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4196..4214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4210..4257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4478..5137)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5052..5071
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5138..5240)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
       61 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      121 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      181 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      241 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      301 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
      361 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
      421 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
      481 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
      541 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
      601 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
      661 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
      721 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
      781 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca
      841 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc
      901 atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      961 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1021 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1081 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1141 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1201 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1261 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     1321 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1381 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1441 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1501 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1561 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1621 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1681 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1741 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1801 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1861 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1921 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1981 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2041 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2101 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2161 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     2221 aattaatacg actcactata ggggaattgt gagcggataa caattcccct gtagaaataa
     2281 ttttgtttaa ctttaataag gagatatacc atgggcagca gccatcacca tcatcaccac
     2341 agccaggatc cggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     2401 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     2461 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     2521 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     2581 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     2641 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     2701 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     2761 ggccccgtaa tgcagaagaa gactatgggc tgggaggcct cctccgagcg gatgtacccc
     2821 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     2881 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     2941 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     3001 cagtacgaac gcgccgaggg ccgccactcc accggcggca tgaagcttgc catggtgaaa
     3061 tcagagaact tgtctaccgg gaatgcgttg attgataacg aactggatat ccgtggttta
     3121 ttccgcctgc tgtggcgtgg aaaggtgtgg ccgatagcaa tcggcctgct gtttgccgtc
     3181 gtggcattag gctattccta tcttgttaaa caggaatggg gtgcaacatc gattaccgac
     3241 aagccgacgg ttaatatgtt gggcggctat tactcgcagc agcaattcct acgtaacctc
     3301 gacgctcgat cgttttcctc tcctcagcca gaacaaccgt ctatttcggc tggtgcttat
     3361 gatgaattca tcatgcagtt agcggcttac gatacccgcc gcgatttctg gttgcagact
     3421 gactattata agcaacgtct ggaaggtgat gaaaaggctg atgcagcctt gctggatgag
     3481 ctggtcaaca acattcagtt tacggcgcgt gatgacggga aaaaaaccaa tgactccgtg
     3541 aagctggtgg cggaaacctc ctcagattcc aacacgttgc ttcgtcagta tgtggttttt
     3601 gccagtcagc gtgcagccaa ccacttgaat gaagaaatca agggagcctg ggctgccaga
     3661 accattttca tgaagtcgca gattaagcgt caggaagcgg tagcaaaagc gatttacgat
     3721 cgtgaagtcc gcagcgttga actggcgctg aaaatcgcac aacagcaagg tattagccgt
     3781 agccagacgg atacgccagc agatgaaatc cccgcatcgg aaatgttcct gctcggcagg
     3841 ccgatgcttc aagcccgact ggaaacgttg cagaccagcg gcccgcatta tgaactggat
     3901 tacgatcaaa atcgcgcgat gttggcgacg ctgaacgttg gcccaacgct tgaggccagt
     3961 ttccagacgt atcgttattt gcgtacaccg gaagaaccgg tgaaacgcga cagcccgcgc
     4021 cgggcattcc tgatggtgat gtggggtgcg attggtgtgc tagtgggagc gggtgttgcc
     4081 ctggctcgcc gtctgcgtta gctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa
     4141 cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct
     4201 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     4261 aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     4321 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg
     4381 aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac
     4441 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
     4501 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat
     4561 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca
     4621 tagtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga
     4681 aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat
     4741 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
     4801 aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg
     4861 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga
     4921 actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt
     4981 gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat
     5041 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
     5101 tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa
     5161 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     5221 aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg
     5281 tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc acaggtattt
     5341 attcggcgca aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt
     5401 ttttgaggtg ctccagtggc ttctgtttct atcagctgtc cctcctgttc agctactgac
     5461 ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag tgtatact
//
