LOCUS       sequence-260191-        3482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro expression of CRM197 with C terminal 4xDQNAT glycosylation 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3482)
  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 3482)
  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: pJL1-CRM197-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..3482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             98..120
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             137..709
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     CDS             1826..1843
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1898..1916)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1912..1959
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1977..2792)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2700..2719
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2784..2803
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(2914..3482,1..20)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3403..3422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gggggcggag cctatggaaa aacgccagca acgcgatccc gcgaaattaa tacgactcac
       61 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga
      121 tatacatatg ggcgctgatg atgttgttga ttcttctaaa tcttttgtga tggaaaactt
      181 ttcttcgtac cacgggacta aacctggtta tgtagattcc attcaaaaag gtatacaaaa
      241 gccaaaatct ggtacacaag gaaattatga cgatgattgg aaagaatttt atagtaccga
      301 caataaatac gacgctgcgg gatactctgt agataatgaa aacccgctct ctggaaaagc
      361 tggaggcgtg gtcaaagtga cgtatccagg actgacgaag gttctcgcac taaaagtgga
      421 taatgccgaa actattaaga aagagttagg tttaagtctc actgaaccgt tgatggagca
      481 agtcggaacg gaagagttta tcaaaaggtt cggtgatggt gcttcgcgtg tagtgctcag
      541 ccttcccttc gctgagggga gttctagcgt tgaatatatt aataactggg aacaggcgaa
      601 agcgttaagc gtagaacttg agattaattt tgaaacccgt ggaaaacgtg gccaagatgc
      661 gatgtatgag tatatggctc aagcctgtgc aggaaatcgt gtcaggcgat cagtaggtag
      721 ctcattgtca tgcataaatc ttgattggga tgtcataagg gataaaacta agacaaagat
      781 agagtctttg aaagagcatg gccctatcaa aaataaaatg agcgaaagtc ccaataaaac
      841 agtatctgag gaaaaagcta aacaatacct agaagaattt catcaaacgg cattagagca
      901 tcctgaattg tcagaactta aaaccgttac tgggaccaat cctgtattcg ctggggctaa
      961 ctatgcggcg tgggcagtaa acgttgcgca agttatcgat agcgaaacag ctgataattt
     1021 ggaaaagaca actgctgctc tttcgatact tcctggtatc ggtagcgtaa tgggcattgc
     1081 agacggtgcc gttcaccaca atacagaaga gatagtggca caatcaatag ctttatcatc
     1141 tttaatggtt gctcaagcta ttccattggt aggagagcta gttgatattg gtttcgctgc
     1201 atataatttt gtagagagta ttatcaattt atttcaagta gttcataatt cgtataatcg
     1261 tcccgcgtat tctccggggc ataaaacgca accatttctt catgacgggt atgctgtcag
     1321 ttggaacact gttgaagatt cgataatccg aactggtttt caaggggaga gtgggcacga
     1381 cataaaaatt actgctgaaa ataccccgct tccaatcgcg ggtgtcctac taccgactat
     1441 tcctggaaag ctggacgtta ataagtccaa gactcatatt tccgtaaatg gtcggaaaat
     1501 aaggatgcgt tgcagagcta tagacggtga tgtaactttt tgtcgcccta aatctcctgt
     1561 ttatgttggt aatggtgtgc atgcgaatct tcacgtggca tttcacagaa gcagctcgga
     1621 gaaaattcat tctaatgaaa tttcatcgga ttccataggc gttcttgggt accagaaaac
     1681 agtagatcac accaaggtta attctaagct atcgctattt tttgaaatca aaagcctcga
     1741 ggatcagaac gcgaccggcg gtgaccaaaa tgccacaggt ggcgatcaaa acgccaccgg
     1801 cggtgaccag aatgcgacag tcgaccatca ccatcatcac cattaagtcg accggctgct
     1861 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1921 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaagccaa ttctgattag
     1981 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2041 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2101 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2161 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2221 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     2281 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2341 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2401 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2461 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     2521 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2581 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2641 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     2701 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcttc
     2761 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2821 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     2881 ttttgagaca caacgtgaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2941 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3001 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3061 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3121 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3181 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3241 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3301 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3361 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3421 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3481 ag
//
