LOCUS       sequence-261736-        5617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of H. influenzae protein D with C terminal 
            4xDQNAT glycosylation sites in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5617)
  AUTHORS   Stark JC, Jaroentomeechai T, Moeller TD, Hershewe JM, Warfel KF, 
            Moricz BS, Martini AM, Dubner RS, Hsu KJ, Stevenson TC, Jones BD, 
            DeLisa MP, Jewett MC
  TITLE     On-demand biomanufacturing of protective conjugate vaccines.
  JOURNAL   Sci Adv. 2021 Feb 3;7(6). pii: 7/6/eabe9444. doi: 
            10.1126/sciadv.abe9444. Print 2021 Feb.
  PUBMED    33536221
REFERENCE   2  (bases 1 to 5617)
  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: "Sci Adv."; 
            date: "2021-02-3"; volume: "7(6). pii: 7/6/eabe9444. doi"; pages: " 
            10.1126/sciadv.abe9444. Print 2021 Feb"
COMMENT     Sequence Label: pTrc99S-ssDsbA-PD-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..5617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1245..1250,1251..1267,1268..1274)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1282..1298
                     /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)."
     primer_bind     1287..1309
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             2465..2482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2524..2541)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2694..2780
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2872..2899
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2918..3009
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3335..4126
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4370..4958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4859..4878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5112..5129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5144..5284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5210..5229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5370..5392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5470..5547
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             join(5548..5617,1..1013)
                     /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     complement(5567..5586)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
       61 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
      121 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
      181 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
      241 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
      301 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
      361 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
      421 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
      481 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
      541 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
      601 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
      661 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
      721 ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc
      781 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
      841 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
      901 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
      961 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     1021 gcaattaatg taagttagcg cgaattgatc tggtttgaca gcttatcatc gactgcacgg
     1081 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     1141 taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg
     1201 cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     1261 cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga aacagaccat
     1321 gggcatgaaa aagatttggc tggcgctggc tggtttagtt ttagcgttta gcgcatcggc
     1381 gtctagaggc cggctcggcc ttaagtcaga caagatcatt atcgcgcatc gcggtgcctc
     1441 cggctacttg ccggagcata ctttggagag caaagcctta gcatttgccc aacaagcgga
     1501 ttacttggaa caagacttgg ccatgactaa ggatgggcgt ctggtggtga ttcacgacca
     1561 ttttcttgat ggtcttacgg atgtagctaa gaagtttccg catcgccatc gtaaggacgg
     1621 gcgctattat gtaatcgatt ttaccttgaa ggaaattcaa agcttggaga tgaccgaaaa
     1681 ctttgagacg aaggatggga agcaagcgca agtctacccg aatcgttttc ccttatggaa
     1741 aagccatttt cgcatccata cttttgagga tgagattgag tttatccaag ggctggaaaa
     1801 gtctactggc aagaaagttg ggatctatcc tgagatcaag gcaccgtggt ttcatcatca
     1861 aaacggtaag gacatcgccg ctgagacctt aaaagtactg aagaagtatg gctacgataa
     1921 aaagactgac atggtttacc ttcaaacgtt cgattttaat gagttgaaac gtattaaaac
     1981 agaattattg ccccagatgg ggatggattt gaagttggtt cagttaatcg cctacaccga
     2041 ctggaaggaa acgcaggaga aggatcccaa aggatactgg gtgaattata attatgattg
     2101 gatgttcaag ccaggagcaa tggctgaagt agtaaaatat gcggatggtg taggccccgg
     2161 ctggtatatg cttgtgaaca aagaagaaag caagcctgat aatatcgtct atactccctt
     2221 agtcaaagaa ctggcccaat ataacgtaga agttcaccct tacaccgtcc gtaaggacgc
     2281 cttaccggag ttttttactg acgtcaatca aatgtatgat gccttgctga acaagagtgg
     2341 agcgacaggc gtcttcaccg actttccaga cactctcgag gatcagaacg cgaccggcgg
     2401 tgaccaaaat gccacaggtg gcgatcaaaa cgccaccggc ggtgaccaga atgcgacagt
     2461 cgaccatcac catcatcacc attaaaagct tggctgtttt ggcggatgag agaagatttt
     2521 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     2581 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     2641 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     2701 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     2761 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     2821 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     2881 tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat
     2941 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3001 aggaagagta tgagtattca acatttccgt gtcgcccgac gtcgatatcc ggatgaaggc
     3061 acgaacccag tggacataag cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg
     3121 ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt
     3181 tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca
     3241 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca
     3301 gcagggcagt cgccctaaaa caaagttaaa catcatgagg gaagcggtga tcgccgaagt
     3361 atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct
     3421 ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga
     3481 tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga
     3541 ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac
     3601 cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt
     3661 tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat
     3721 tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc
     3781 ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac
     3841 cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac
     3901 gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc
     3961 cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca
     4021 ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt
     4081 tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataatgtc taacaattcg
     4141 ttcaagccga cggatatcct cgagctgaga taggtgcctc actgattaag cattggtaac
     4201 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4261 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4321 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4381 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4441 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4501 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4561 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4621 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4681 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4741 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4801 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4861 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4921 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4981 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5041 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5101 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5161 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     5221 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5281 tgcgccccga cacccgccaa cacccgctga ctcgccctga cgggcttgtc tgctcccggc
     5341 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5401 gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc gcgaaggcga agcggcatgc
     5461 atttacgttg acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg
     5521 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     5581 tatgccggtg tctcttatca gaccgtttcc cgcgtgg
//
