LOCUS       sequence-263353-        6250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of CRM197 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 6250)
  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 6250)
  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-CRM197-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..6250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(67..72,73..89,90..96)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    104..120
                     /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     109..131
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             231..803
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     CDS             1920..1937
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1979..1996)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2149..2235
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2327..2354
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2373..2464
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2790..3581
                     /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      3825..4413
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4314..4333
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4567..4584
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4599..4739
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4665..4684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4825..4847
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4925..5002
                     /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             5003..6085
                     /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(5022..5041)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
       61 gagctgttga caattaatca tccggctcgt ataatgtgtg gaattgtgag cggataacaa
      121 tttcacacag gaaacagacc atgggcatga aaaagatttg gctggcgctg gctggtttag
      181 ttttagcgtt tagcgcatcg gcgtctagag gccggctcgg ccttggcgct gatgatgttg
      241 ttgattcttc taaatctttt gtgatggaaa acttttcttc gtaccacggg actaaacctg
      301 gttatgtaga ttccattcaa aaaggtatac aaaagccaaa atctggtaca caaggaaatt
      361 atgacgatga ttggaaagaa ttttatagta ccgacaataa atacgacgct gcgggatact
      421 ctgtagataa tgaaaacccg ctctctggaa aagctggagg cgtggtcaaa gtgacgtatc
      481 caggactgac gaaggttctc gcactaaaag tggataatgc cgaaactatt aagaaagagt
      541 taggtttaag tctcactgaa ccgttgatgg agcaagtcgg aacggaagag tttatcaaaa
      601 ggttcggtga tggtgcttcg cgtgtagtgc tcagccttcc cttcgctgag gggagttcta
      661 gcgttgaata tattaataac tgggaacagg cgaaagcgtt aagcgtagaa cttgagatta
      721 attttgaaac ccgtggaaaa cgtggccaag atgcgatgta tgagtatatg gctcaagcct
      781 gtgcaggaaa tcgtgtcagg cgatcagtag gtagctcatt gtcatgcata aatcttgatt
      841 gggatgtcat aagggataaa actaagacaa agatagagtc tttgaaagag catggcccta
      901 tcaaaaataa aatgagcgaa agtcccaata aaacagtatc tgaggaaaaa gctaaacaat
      961 acctagaaga atttcatcaa acggcattag agcatcctga attgtcagaa cttaaaaccg
     1021 ttactgggac caatcctgta ttcgctgggg ctaactatgc ggcgtgggca gtaaacgttg
     1081 cgcaagttat cgatagcgaa acagctgata atttggaaaa gacaactgct gctctttcga
     1141 tacttcctgg tatcggtagc gtaatgggca ttgcagacgg tgccgttcac cacaatacag
     1201 aagagatagt ggcacaatca atagctttat catctttaat ggttgctcaa gctattccat
     1261 tggtaggaga gctagttgat attggtttcg ctgcatataa ttttgtagag agtattatca
     1321 atttatttca agtagttcat aattcgtata atcgtcccgc gtattctccg gggcataaaa
     1381 cgcaaccatt tcttcatgac gggtatgctg tcagttggaa cactgttgaa gattcgataa
     1441 tccgaactgg ttttcaaggg gagagtgggc acgacataaa aattactgct gaaaataccc
     1501 cgcttccaat cgcgggtgtc ctactaccga ctattcctgg aaagctggac gttaataagt
     1561 ccaagactca tatttccgta aatggtcgga aaataaggat gcgttgcaga gctatagacg
     1621 gtgatgtaac tttttgtcgc cctaaatctc ctgtttatgt tggtaatggt gtgcatgcga
     1681 atcttcacgt ggcatttcac agaagcagct cggagaaaat tcattctaat gaaatttcat
     1741 cggattccat aggcgttctt gggtaccaga aaacagtaga tcacaccaag gttaattcta
     1801 agctatcgct attttttgaa atcaaaagcc tcgaggatca gaacgcgacc ggcggtgacc
     1861 aaaatgccac aggtggcgat caaaacgcca ccggcggtga ccagaatgcg acagtcgacc
     1921 atcaccatca tcaccattaa aagcttggct gttttggcgg atgagagaag attttcagcc
     1981 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     2041 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
     2101 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga
     2161 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2221 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     2281 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     2341 acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa
     2401 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2461 gagtatgagt attcaacatt tccgtgtcgc ccgacgtcga tatccggatg aaggcacgaa
     2521 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
     2581 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     2641 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     2701 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     2761 gcagtcgccc taaaacaaag ttaaacatca tgagggaagc ggtgatcgcc gaagtatcga
     2821 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     2881 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     2941 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     3001 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     3061 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     3121 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     3181 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     3241 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     3301 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     3361 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     3421 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     3481 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     3541 actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctaaca attcgttcaa
     3601 gccgacggat atcctcgagc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3661 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3721 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3781 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3841 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3901 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3961 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4021 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4081 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4141 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4201 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4261 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4321 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4381 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4441 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4501 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4561 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4621 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     4681 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4741 cccgacaccc gccaacaccc gctgactcgc cctgacgggc ttgtctgctc ccggcatccg
     4801 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4861 caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta
     4921 cgttgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     4981 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     5041 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     5101 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     5161 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     5221 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     5281 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     5341 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     5401 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     5461 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     5521 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     5581 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     5641 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     5701 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     5761 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     5821 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     5881 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     5941 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     6001 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6061 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag cgcgaattga
     6121 tctggtttga cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc
     6181 catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca
     6241 aggcgcactc
//
