LOCUS       sequence-263351-        5998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of tetanus toxin fragment C 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 5998)
  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 5998)
  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-TTc-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..5998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(195..200,201..217,218..224)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    232..248
                     /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     237..259
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1796..1813
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1855..1872)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2025..2111
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2203..2230
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2249..2340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2666..3457
                     /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      3701..4289
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4190..4209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4443..4460
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4475..4615
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4541..4560)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4701..4723
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4801..4878
                     /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             4879..5961
                     /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(4898..4917)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tggtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca
       61 tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag
      121 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
      181 tctgaaatga gctgttgaca attaatcatc cggctcgtat aatgtgtgga attgtgagcg
      241 gataacaatt tcacacagga aacagaccat gggcatgaaa aagatttggc tggcgctggc
      301 tggtttagtt ttagcgttta gcgcatcggc gtctagaggc cggctcggcc ttaagaatct
      361 ggattgctgg gtggacaatg aagaagacat cgacgtgatt cttaaaaaga gtacgatcct
      421 taacttggat attaacaatg acattatttc agacatctct ggtttcaata gttcagtcat
      481 tacatatccg gatgcccaac tggttccagg aatcaatgga aaggcgatcc accttgtgaa
      541 caatgagtct agcgaggtga ttgtccataa ggctatggac atcgaataca acgatatgtt
      601 taacaacttc acagtatcgt tttggctgcg tgtaccaaaa gtctcagctt ctcatttgga
      661 acaatacgat acaaatgaat actccatcat cagctctatg aagaagtata gcctttcgat
      721 cggcagtggt tggtccgtgt ccttaaaagg taataactta atctggacat taaaagattc
      781 agccggcgaa gtccgtcaga tcactttccg cgacctttcc gataaattta acgcttactt
      841 agcaaacaaa tgggtattca ttaccatcac aaacgaccgt ttgagctctg ccaatttgta
      901 cattaacggt gttttaatgg gtagcgcaga gatcacgggc cttggggcta tccgtgagga
      961 caataatatc acacttaagc tggatcgctg caataacaac aatcaatatg tgagcatcga
     1021 caaatttcgc attttttgta aggctcttaa ccccaaggag atcgaaaagt tatatacgag
     1081 ttacctgagc attacctttc tgcgcgactt ttgggggaat ccgttacgct atgacacaga
     1141 atattattta atccctgtgg catactcttc gaaagacgtc cagctgaaaa acattaccga
     1201 ctatatgtac ttaacgaacg ctccaagcta cacgaatgga aaattaaaca tctattaccg
     1261 tcgtttatat tctggtctga aatttatcat taaacgctat acacccaata atgagattga
     1321 ttcgttcgtc cgttccggtg acttcattaa gttgtatgtt tcgtacaaca acaacgaaca
     1381 tattgttggg taccccaaag atggaaacgc tttcaataat ttggaccgca tcttacgtgt
     1441 aggctacaac gctccaggga tccctcttta caaaaaaatg gaagcggtaa aacttcgcga
     1501 cttaaaaact tattcagtcc agcttaagct ttatgacgat aaagatgcct cgcttggcct
     1561 tgttgggact cataacggcc aaatcggtaa tgaccccaac cgcgacatcc ttattgcatc
     1621 caattggtac tttaatcacc tgaaggataa gactctgact tgcgattggt acttcgtacc
     1681 gactgacgaa gggtggacga acgatctcga ggatcagaac gcgaccggcg gtgaccaaaa
     1741 tgccacaggt ggcgatcaaa acgccaccgg cggtgaccag aatgcgacag tcgaccatca
     1801 ccatcatcac cattaaaagc ttggctgttt tggcggatga gagaagattt tcagcctgat
     1861 acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag
     1921 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     1981 tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     2041 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     2101 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     2161 gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg
     2221 atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg
     2281 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2341 atgagtattc aacatttccg tgtcgcccga cgtcgatatc cggatgaagg cacgaaccca
     2401 gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa
     2461 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
     2521 ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg
     2581 ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag
     2641 tcgccctaaa acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca
     2701 actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca
     2761 tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt
     2821 tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga
     2881 aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt
     2941 gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg
     3001 gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc
     3061 tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga
     3121 actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct
     3181 atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg
     3241 catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc
     3301 aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct
     3361 tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta
     3421 cgtgaaaggc gagatcacca aggtagtcgg caaataatgt ctaacaattc gttcaagccg
     3481 acggatatcc tcgagctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3541 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3601 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3661 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3721 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3781 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3841 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3901 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3961 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4021 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4081 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4141 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4201 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4261 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4321 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4381 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4441 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4501 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     4561 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4621 acacccgcca acacccgctg actcgccctg acgggcttgt ctgctcccgg catccgctta
     4681 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4741 gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg catttacgtt
     4801 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     4861 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     4921 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     4981 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     5041 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     5101 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     5161 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     5221 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     5281 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     5341 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     5401 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     5461 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     5521 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     5581 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     5641 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     5701 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     5761 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     5821 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     5881 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5941 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg aattgatc
//
