LOCUS       sequence-389774-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of C-terminally distrep tag II-mCherry-tagged PglL from 
            Neisseria meningitidis in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  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 6566)
  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: pPglL-strep-mCherry
FEATURES             Location/Qualifiers
     source          1..6566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..81)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(198..743)
                     /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(278..297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(968..2050)
                     /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     2012..2031
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2051..2128)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        2174..2192
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2193..2217
                     /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             4082..4102
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4106..4129
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4166..4189
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(4338..4356)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4606..4625
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        4960..4978
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4979..5003
                     /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             5112..5156
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5194..5212)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5208..5255
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5476..6135)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6050..6069
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6136..6238)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc
       61 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
      121 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
      181 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
      241 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      301 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
      361 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
      421 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
      481 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
      541 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
      601 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
      661 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
      721 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga
      781 tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt
      841 tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
      901 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
      961 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1021 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1081 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1141 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1201 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     1261 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1321 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1381 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1441 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1501 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1561 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1621 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     1681 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     1741 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     1801 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     1861 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     1921 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     1981 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2041 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2101 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     2161 tgcattagga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     2221 gtagaaataa ttttgtttaa ctttaataag gagatatacc atggcacccg ctgaaacgac
     2281 cgtatccggc gcgcaccccg ccgccaaact gccgatttac atcctgccct gcttcctttg
     2341 gataggcatc gtccccttta ccttcgcgct caaactgaaa ccgtcgcccg acttttacca
     2401 cgatgccgcc gccgcagccg gcctgattgt cctgttgttc ctcacggcag gaaaaaaact
     2461 gtttgatgtc aaaatccccg ccatcagctt ccttctgttt gcaatggcgg cgttttggta
     2521 tcttcaggca cgcctgatga acctgattta ccccggtatg aacgacatcg tctcttggat
     2581 tttcatcttg ctcgccgtca gcgcgtgggc ctgccggagc ttggtcgcac acttcggaca
     2641 agaacgcatc gtgaccctgt ttgcctggtc gctgcttatc ggctccctgc ttcaatcctg
     2701 catcgtcgtc atccagtttg ccggctggga agacacccct ctgtttcaaa acatcatcgt
     2761 ttacagcggg caaggcgtaa tcggacacat cgggcagcgc aacaacctcg gacactacct
     2821 catgtggggc atactcgccg ccgcctacct caacggacaa cgaaaaatcc ccgccgccct
     2881 cggcgtaatc tgcctgatta tgcagaccgc cgttttaggt ttggtcaact cgcgcaccat
     2941 cttgacctac atagccgcca tcgccctcat ccttcccttc tggtatttcc gttcggacaa
     3001 atccaacagg cggacgatgc tcggcatagc cgcagccgta ttccttaccg cgctgttcca
     3061 attttccatg aacaccattc tggaaacctt tactggcatc cgctacgaaa ctgccgtcga
     3121 acgcgtcgcc aacggcggtt tcacagactt gccgcgccaa atcgaatgga ataaagccct
     3181 tgccgccttc cagtccgccc cgatattcgg gcacggctgg aacagttttg cccaacaaac
     3241 cttcctcatc aatgccgaac agcacaacat atacgacaac ctcctcagca acttgttcac
     3301 ccattcccac aacatcgtcc tccaactcct tgcagagatg ggaatcagcg gcacgcttct
     3361 ggttgccgca accctgctga cgggcattgc cgggctgctt aaacgccccc tgacccccgc
     3421 atcgcttttc ctaatctgca cgcttgccgt cagtatgtgc cacagtatgc tcgaatatcc
     3481 tttgtggtat gtctatttcc tcatcccttt cggactgatg ctcttcctgt cccccgcaga
     3541 ggcttcagac ggcatcgcct tcaaaaaagc cgccaatctc ggcatactga ccgcctccgc
     3601 cgccatattc gcaggattgc tgcacttgga ctggacatac acccggctgg ttaacgcctt
     3661 ttcccccgcc actgacgaca gtgccaaaac cctcaaccgg aaaatcaacg agttgcgcta
     3721 tatttccgca aacagtccga tgctgtcctt ttatgccgac ttctccctcg taaacttcgc
     3781 cctgccggaa taccccgaaa cccagacttg ggcggaagaa gcaaccctca aatcactaaa
     3841 ataccgcccc cactccgcca cctaccgcat cgccctctac ctgatgcggc aaggcaaagt
     3901 tgcagaagca aaacaatgga tgcgggcgac acagtcctat tacccctacc tgatgccccg
     3961 atacgccgac gaaatccgca aactgcccgt atgggcgccg ctgctacccg aactgctcaa
     4021 agactgcaaa gccttcgccg ccgcgcccgg tcatccggaa gcaaaaccct gcaaactcga
     4081 ggaaaacctg tattttcagg gcgcgtggtc ccatcctcag ttcgagaaag gcgcgggttc
     4141 aggtgctggc agtggagcgt ccgcgtggag tcatccgcaa ttcgaaaaag aattcgtgag
     4201 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     4261 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     4321 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     4381 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     4441 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     4501 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     4561 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     4621 gaagactatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     4681 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     4741 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     4801 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     4861 gggccgccac tccaccggcg gcatgtagaa gcttgcggcc gcataatgct taagtcgaac
     4921 agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg
     4981 aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata
     5041 tacatatggc agatctcaat tggatatcgg ccggccacgc gatcgctgac gtcggtaccc
     5101 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     5161 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     5221 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
     5281 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     5341 ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc
     5401 attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata
     5461 actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     5521 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     5581 catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg gggcgaagaa
     5641 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     5701 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     5761 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     5821 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     5881 ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag
     5941 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     6001 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     6061 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     6121 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     6181 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     6241 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     6301 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg
     6361 tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt
     6421 ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa
     6481 gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga
     6541 gggtgtcagt gaagtgcttc atgtgg
//
