LOCUS       sequence-389757-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of C-terminally distrep tag II-mClover3-tagged PglL from 
            Neisseria meningitidis in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7232)
  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 7232)
  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-mClover3
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..102
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(282..1160)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1187..1471
                     /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     1394..1413
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3327..3347
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3351..3374
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3411..3434
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             join(3441..3443,3444..4157)
                     /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(3483..3504)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4091..4112
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4179..4199
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4203..4232
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(4276..4293)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4446..4532
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4624..4651
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4670..4761
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4762..4830,4831..5622)
                     /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(4980..4999)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5664..6119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5751..5770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5961..5982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      6230..6818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6719..6738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6972..6989
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7155..7177
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
       61 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
      121 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca tgtttgacag
      181 cttatcatcg atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta
      241 tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct
      301 acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat
      361 tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg
      421 gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg
      481 cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc
      541 gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg
      601 ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta
      661 atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa
      721 tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc
      781 tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc
      841 cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc
      901 tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt
      961 cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg
     1021 agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc
     1081 tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga
     1141 tcattttgcg cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt
     1201 ccatattgca tcagacattg ccgtcactgc gtcttttact ggctcttctc gctaaccaaa
     1261 ccggtaaccc cgcttattaa aagcattctg taacaaagcg ggaccaaagc catgacaaaa
     1321 acgcgtaaca aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc
     1381 gtcacacttt gctatgccat agcattttta tccataagat tagcggatct tacctgacgc
     1441 tttttatcgc aactctctac tgtttctcca tacccgtttt tttgggctag caggaggaat
     1501 acaccatggc acccgctgaa acgaccgtat ccggcgcgca ccccgccgcc aaactgccga
     1561 tttacatcct gccctgcttc ctttggatag gcatcgtccc ctttaccttc gcgctcaaac
     1621 tgaaaccgtc gcccgacttt taccacgatg ccgccgccgc agccggcctg attgtcctgt
     1681 tgttcctcac ggcaggaaaa aaactgtttg atgtcaaaat ccccgccatc agcttccttc
     1741 tgtttgcaat ggcggcgttt tggtatcttc aggcacgcct gatgaacctg atttaccccg
     1801 gtatgaacga catcgtctct tggattttca tcttgctcgc cgtcagcgcg tgggcctgcc
     1861 ggagcttggt cgcacacttc ggacaagaac gcatcgtgac cctgtttgcc tggtcgctgc
     1921 ttatcggctc cctgcttcaa tcctgcatcg tcgtcatcca gtttgccggc tgggaagaca
     1981 cccctctgtt tcaaaacatc atcgtttaca gcgggcaagg cgtaatcgga cacatcgggc
     2041 agcgcaacaa cctcggacac tacctcatgt ggggcatact cgccgccgcc tacctcaacg
     2101 gacaacgaaa aatccccgcc gccctcggcg taatctgcct gattatgcag accgccgttt
     2161 taggtttggt caactcgcgc accatcttga cctacatagc cgccatcgcc ctcatccttc
     2221 ccttctggta tttccgttcg gacaaatcca acaggcggac gatgctcggc atagccgcag
     2281 ccgtattcct taccgcgctg ttccaatttt ccatgaacac cattctggaa acctttactg
     2341 gcatccgcta cgaaactgcc gtcgaacgcg tcgccaacgg cggtttcaca gacttgccgc
     2401 gccaaatcga atggaataaa gcccttgccg ccttccagtc cgccccgata ttcgggcacg
     2461 gctggaacag ttttgcccaa caaaccttcc tcatcaatgc cgaacagcac aacatatacg
     2521 acaacctcct cagcaacttg ttcacccatt cccacaacat cgtcctccaa ctccttgcag
     2581 agatgggaat cagcggcacg cttctggttg ccgcaaccct gctgacgggc attgccgggc
     2641 tgcttaaacg ccccctgacc cccgcatcgc ttttcctaat ctgcacgctt gccgtcagta
     2701 tgtgccacag tatgctcgaa tatcctttgt ggtatgtcta tttcctcatc cctttcggac
     2761 tgatgctctt cctgtccccc gcagaggctt cagacggcat cgccttcaaa aaagccgcca
     2821 atctcggcat actgaccgcc tccgccgcca tattcgcagg attgctgcac ttggactgga
     2881 catacacccg gctggttaac gccttttccc ccgccactga cgacagtgcc aaaaccctca
     2941 accggaaaat caacgagttg cgctatattt ccgcaaacag tccgatgctg tccttttatg
     3001 ccgacttctc cctcgtaaac ttcgccctgc cggaataccc cgaaacccag acttgggcgg
     3061 aagaagcaac cctcaaatca ctaaaatacc gcccccactc cgccacctac cgcatcgccc
     3121 tctacctgat gcggcaaggc aaagttgcag aagcaaaaca atggatgcgg gcgacacagt
     3181 cctattaccc ctacctgatg ccccgatacg ccgacgaaat ccgcaaactg cccgtatggg
     3241 cgccgctgct acccgaactg ctcaaagact gcaaagcctt cgccgccgcg cccggtcatc
     3301 cggaagcaaa accctgcaaa ctcgaggaaa acctgtattt tcagggcgcg tggtcccatc
     3361 ctcagttcga gaaaggcgcg ggttcaggtg ctggcagtgg agcgtccgcg tggagtcatc
     3421 cgcaattcga aaaagaattc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     3481 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg
     3541 agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     3601 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc ttcagccgct
     3661 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     3721 aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt
     3781 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3841 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccactacgtc tatatcacgg
     3901 ccgacaagca gaagaactgc atcaaggcta acttcaagat ccgccacaac gttgaggacg
     3961 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     4021 tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac cccaacgaga
     4081 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggattaca catggcatgg
     4141 acgagctgta caagtagaag cttgcggccg cactcgagga aaacctgtat tttcagggcg
     4201 cgcaccacca ccaccaccat caccatcacc attgataacg cttggctgtt ttggcggatg
     4261 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
     4321 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     4381 gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
     4441 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     4501 tgtcagtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     4561 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     4621 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt
     4681 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4741 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4801 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4861 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4921 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4981 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     5041 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5101 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5161 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5221 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5281 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cattgcgcaa actattaact
     5341 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5401 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5461 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5521 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5581 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5641 tcatatatac tttagattga tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5701 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5761 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5821 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5881 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5941 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     6001 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     6061 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaattta
     6121 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6181 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6241 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6301 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6361 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6421 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6481 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6541 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6601 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6661 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6721 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6781 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6841 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     6901 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     6961 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     7021 tccttacgca tctgtgcggt atttcacacc gcatagggtc atggctgcgc cccgacaccc
     7081 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     7141 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     7201 cgcgaggcag aaggagatgg cgcccaacag tc
//
