LOCUS       sequence-263348-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of N. meningitidis PorA 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 5764)
  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 5764)
  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-PorA-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      116..202
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      294..321
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        340..431
                     /gene="bla"
                     /label=AmpR promoter
     CDS             757..1548
                     /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      1792..2380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2281..2300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2534..2551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2566..2706
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2632..2651)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2792..2814
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2892..2969
                     /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             2970..4052
                     /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(2989..3008)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4284..4289,4290..4306,4307..4313)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4321..4337
                     /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     4326..4348
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             5651..5668
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5710..5727)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
       61 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
      121 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      181 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
      241 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
      301 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
      361 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      421 aaaggaagag tatgagtatt caacatttcc gtgtcgcccg acgtcgatat ccggatgaag
      481 gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg
      541 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
      601 gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag
      661 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg
      721 cagcagggca gtcgccctaa aacaaagtta aacatcatga gggaagcggt gatcgccgaa
      781 gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg
      841 ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt
      901 gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac
      961 gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc
     1021 accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa
     1081 tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac
     1141 attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca
     1201 gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa
     1261 accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt
     1321 acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct
     1381 gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga
     1441 caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa
     1501 tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataatg tctaacaatt
     1561 cgttcaagcc gacggatatc ctcgagctga gataggtgcc tcactgatta agcattggta
     1621 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     1681 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     1741 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1801 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1861 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1921 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1981 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2041 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2101 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2161 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2221 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2281 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2341 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2401 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2461 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2521 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2581 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     2641 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     2701 gctgcgcccc gacacccgcc aacacccgct gactcgccct gacgggcttg tctgctcccg
     2761 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2821 ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat
     2881 gcatttacgt tgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc
     2941 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     3001 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     3061 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     3121 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     3181 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     3241 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     3301 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     3361 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     3421 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     3481 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     3541 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     3601 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     3661 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     3721 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     3781 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     3841 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     3901 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     3961 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4021 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagcgc
     4081 gaattgatct ggtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc
     4141 aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg
     4201 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
     4261 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa
     4321 ttgtgagcgg ataacaattt cacacaggaa acagaccatg ggcatgaaaa agatttggct
     4381 ggcgctggct ggtttagttt tagcgtttag cgcatcggcg tctagaggcc ggctcggcct
     4441 tgacgtcagt ttgtatggag agattaaggc aggggttgag gggcgcaatt accaacttca
     4501 acttacggaa gcgcaggcag ctaatggtgg ggcgagtggc caggtcaaag tcacgaaggt
     4561 tactaaggca aagtcccgca ttcgtacgaa gatttccgac tttggctcat ttattggttt
     4621 caagggttcc gaggacttag gcgatggctt gaaagcagtt tggcagcttg agcaagatgt
     4681 gagtgtggcg ggagggggcg ctacccagtg gggtaaccgt gagtctttta tcgggttagc
     4741 aggcgagttc ggaactttac gtgccgggcg cgtagcaaac cagtttgacg atgcctccca
     4801 ggccatcgac ccctgggatt ctaataatga cgtagcaagc caactgggaa tttttaagcg
     4861 ccacgatgat atgcctgtct ccgttcgtta cgacagtccg gagtttagcg ggttctcggg
     4921 atctgtgcaa ttcgttccga tccaaaacag taaatctgcc tatacaccgg catactatac
     4981 aaaaaacaca aataataact tgaccttagt accagccgtg gttggaaagc caggttctga
     5041 tgtatattat gcgggactga actacaaaaa cggagggttc gccggtaact atgcgtttaa
     5101 gtacgcccgc cacgctaacg tcggccgtaa cgcctttgaa ctgttcttga ttgggagcgg
     5161 atcggatcaa gcgaaaggaa cagaccccct gaaaaaccat caagtccacc gtttgaccgg
     5221 aggatatgag gaggggggtt tgaacttggc attagccgcg cagttggact tatcggaaaa
     5281 cggcgacaaa actaagaatt ccactactga aatcgcagct actgcgagct atcgttttgg
     5341 gaatgcagtg cctcgtattt catacgctca cggcttcgat ttcattgagc gcggcaaaaa
     5401 gggagaaaat acgtcctacg accaaattat cgccggtgta gattatgact tctctaagcg
     5461 tactagcgca atcgtgagcg gcgcttggtt gaagcgcaat acgggtatcg ggaattatac
     5521 ccaaatcaat gccgcatctg tcggcttgcg tcacaaattt ctcgaggatc agaacgcgac
     5581 cggcggtgac caaaatgcca caggtggcga tcaaaacgcc accggcggtg accagaatgc
     5641 gacagtcgac catcaccatc atcaccatta aaagcttggc tgttttggcg gatgagagaa
     5701 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     5761 gcct
//
