LOCUS       sequence-261737-        5755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of E. coli MBP 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 5755)
  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 5755)
  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-MBP-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..5755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(211..216,217..233,234..240)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    248..264
                     /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     253..275
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             369..1466
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     HAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1440..1463
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1569..1586
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1628..1645)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1798..1884
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1976..2003
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2022..2113
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2439..3230
                     /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      3474..4062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3963..3982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4216..4233
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4248..4388
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4314..4333)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4474..4496
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4574..4651
                     /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             4652..5734
                     /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(4671..4690)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttagcgcgaa ttgatctggt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
       61 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
      121 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
      181 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatccggc tcgtataatg
      241 tgtggaattg tgagcggata acaatttcac acaggaaaca gaccatgggc atgaaaaaga
      301 tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcgtct agaggccggc
      361 tcggccttaa aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata
      421 acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg
      481 agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg
      541 acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg
      601 aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac
      661 gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata
      721 acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag
      781 aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct
      841 ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca
      901 ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc
      961 tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta
     1021 ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca
     1081 gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt
     1141 tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcgaaag
     1201 agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac
     1261 cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg
     1321 ccgccacaat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg
     1381 ctttctggca tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg
     1441 atgaagccct gaaagacgcg cagactcgta tcaccaagct cgaggatcag aacgcgaccg
     1501 gcggtgacca aaatgccaca ggtggcgatc aaaacgccac cggcggtgac cagaatgcga
     1561 cagtcgacca tcaccatcat caccattaaa agcttggctg ttttggcgga tgagagaaga
     1621 ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc
     1681 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     1741 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     1801 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     1861 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     1921 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     1981 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat
     2041 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2101 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cgacgtcgat atccggatga
     2161 aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta
     2221 tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg
     2281 cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca
     2341 agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta
     2401 cgcagcaggg cagtcgccct aaaacaaagt taaacatcat gagggaagcg gtgatcgccg
     2461 aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt
     2521 tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata
     2581 ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca
     2641 acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag
     2701 tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc
     2761 aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg
     2821 acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc
     2881 cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg
     2941 aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc
     3001 ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg
     3061 ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta
     3121 gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag
     3181 aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa tgtctaacaa
     3241 ttcgttcaag ccgacggata tcctcgagct gagataggtg cctcactgat taagcattgg
     3301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3601 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4081 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4141 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4201 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4261 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     4321 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4381 tggctgcgcc ccgacacccg ccaacacccg ctgactcgcc ctgacgggct tgtctgctcc
     4441 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4501 caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc
     4561 atgcatttac gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
     4621 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     4681 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     4741 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     4801 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     4861 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     4921 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     4981 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     5041 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     5101 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     5161 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     5221 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     5281 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     5341 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     5401 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     5461 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     5521 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     5581 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     5641 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5701 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaag
//
