LOCUS       sequence-252694-        5772 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 5772)
  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 5772)
  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..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     759..776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    791..931
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(857..876)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1017..1039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1117..1194
                     /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             1195..2277
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1214..1233)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2509..2514,2515..2531,2532..2538)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2546..2562
                     /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     2551..2573
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             3876..3893
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3935..3952)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4105..4191
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4283..4310
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4330..4421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4754..5545
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATYDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      841 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
      901 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
      961 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     1021 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa
     1081 ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc
     1141 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     1201 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     1261 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     1321 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     1381 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     1441 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     1501 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     1561 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     1621 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     1681 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     1741 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     1801 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     1861 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     1921 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     1981 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     2041 atcccgccgt caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     2101 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     2161 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2221 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     2281 caacgcaatt aatgtgagtt agcgcgaatt gatctggttt gacagcttat catcgactgc
     2341 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag
     2401 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt
     2461 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat
     2521 catccggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacaga
     2581 ccatgggcat gaaaaagatt tggctggcgc tggctggttt agttttagcg tttagcgcat
     2641 cggcgtctag aggccggctc ggccttgacg tcagtttgta tggagagatt aaggcagggg
     2701 ttgaggggcg caattaccaa cttcaactta cggaagcgca ggcagctaat ggtggggcga
     2761 gtggccaggt caaagtcacg aaggttacta aggcaaagtc ccgcattcgt acgaagattt
     2821 ccgactttgg ctcatttatt ggtttcaagg gttccgagga cttaggcgat ggcttgaaag
     2881 cagtttggca gcttgagcaa gatgtgagtg tggcgggagg gggcgctacc cagtggggta
     2941 accgtgagtc ttttatcggg ttagcaggcg agttcggaac tttacgtgcc gggcgcgtag
     3001 caaaccagtt tgacgatgcc tcccaggcca tcgacccctg ggattctaat aatgacgtag
     3061 caagccaact gggaattttt aagcgccacg atgatatgcc tgtctccgtt cgttacgaca
     3121 gtccggagtt tagcgggttc tcgggatctg tgcaattcgt tccgatccaa aacagtaaat
     3181 ctgcctatac accggcatac tatacaaaaa acacaaataa taacttgacc ttagtaccag
     3241 ccgtggttgg aaagccaggt tctgatgtat attatgcggg actgaactac aaaaacggag
     3301 ggttcgccgg taactatgcg tttaagtacg cccgccacgc taacgtcggc cgtaacgcct
     3361 ttgaactgtt cttgattggg agcggatcgg atcaagcgaa aggaacagac cccctgaaaa
     3421 accatcaagt ccaccgtttg accggaggat atgaggaggg gggtttgaac ttggcattag
     3481 ccgcgcagtt ggacttatcg gaaaacggcg acaaaactaa gaattccact actgaaatcg
     3541 cagctactgc gagctatcgt tttgggaatg cagtgcctcg tatttcatac gctcacggct
     3601 tcgatttcat tgagcgcggc aaaaagggag aaaatacgtc ctacgaccaa attatcgccg
     3661 gtgtagatta tgacttctct aagcgtacta gcgcaatcgt gagcggcgct tggttgaagc
     3721 gcaatacggg tatcgggaat tatacccaaa tcaatgccgc atctgtcggc ttgcgtcaca
     3781 aatttctcga ggatcagaac gcgaccggcg gtgaccaaaa tgccacaggt ggcgatcaaa
     3841 acgccaccgg cggtgaccag aatgcgacag tcgaccatca ccatcatcac cattaaaagc
     3901 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     3961 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     4021 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     4081 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4141 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     4201 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     4261 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     4321 caaactcttt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4381 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4441 gtgtcgcccg acgtcgatat ccggatgaag gcacgaaccc agtggacata agcctcgttc
     4501 ggttcgtaag ctgtaatgca agtagcgtaa ctgccgtcac gcaactggtc cacaaccttg
     4561 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttcttg ttatgacatg
     4621 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
     4681 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
     4741 aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
     4801 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
     4861 cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt
     4921 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
     4981 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
     5041 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
     5101 cattcttgca ggtatcttcg agccagccac gtacgacatt gatctggcta tcttgctgac
     5161 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
     5221 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
     5281 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     5341 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     5401 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     5461 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga
     5521 gatcaccaag gtagtcggca aataatgtct aacaattcgt tcaagccgac ggatatcctc
     5581 gagctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5641 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5701 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5761 accccgtaga aa
//
