LOCUS       sequence-263357-        6013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of tetanus toxin light chain 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 6013)
  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 6013)
  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-TTlight-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..6013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(101..106,107..123,124..130)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    138..154
                     /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     143..165
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1717..1734
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1776..1793)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1946..2032
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2124..2151
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2170..2261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2587..3378
                     /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      3622..4210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4111..4130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4364..4381
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4396..4536
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4462..4481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4622..4644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4722..4799
                     /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             4800..5882
                     /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(4819..4838)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc
       61 gacatcataa cggttctggc aaatattctg aaatgagctg ttgacaatta atcatccggc
      121 tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca gaccatgggc
      181 atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcgtct
      241 agaggccggc tcggccttcc tatcaccatc aataacttcc gctactcaga tccagtgaac
      301 aacgacacta ttattatgat ggaacccccc tattgcaagg gattagatat ctattataaa
      361 gccttcaaga ttaccgatcg tatctggatt gtccccgagc gctacgaatt cgggaccaaa
      421 cccgaagatt ttaatccacc aagcagcttg atcgagggtg cttcggagta ctatgatccc
      481 aactacctgc gtaccgattc ggataaagac cgttttctgc agacgatggt aaaacttttc
      541 aatcgtatta agaataatgt cgcgggcgaa gccttattgg ataagattat caatgcgatt
      601 ccttatcttg gcaacagtta ctcgttactg gacaagtttg atacgaattc aaatagcgtg
      661 agttttaacc tgttggaaca ggacccttca ggcgcgacca ctaaaagcgc aatgttaacc
      721 aacttaatca tttttggacc cggtcccgta ttgaacaaaa atgaggttcg cgggatcgta
      781 ctgcgtgtag acaataagaa ttactttccg tgccgcgatg gctttggctc gattatgcaa
      841 atggcgtttt gcccagagta cgtgccaaca ttcgacaatg tcatcgaaaa tatcacctct
      901 ctgaccatcg gaaagagtaa atatttccaa gatcctgctt tgttgctgat gcatgccctg
      961 atccatgtgt tacatggttt atacggtatg caagtttctt cgcacgaaat tattccctcg
     1021 aaacaggaaa tttacatgca acatacatac ccgattagtg cggaggagct gttcaccttt
     1081 ggcgggcagg acgcgaattt aatctctatt gatatcaaga acgacttata tgaaaagaca
     1141 ttgaacgact acaaagcaat cgcaaacaaa ctgagtcagg tcacctcatg caatgacccg
     1201 aacattgata ttgacagtta taagcagatc taccaacaaa aataccaatt tgataaggat
     1261 tctaatggtc aatatattgt gaacgaggat aagtttcaga ttctttataa tagcatcatg
     1321 tatggtttta ctgagattga gctggggaaa aaattcaaca ttaaaacccg tctttcgtat
     1381 ttttcgatga accacgatcc cgttaaaatt ccgaacttac tggatgacac tatctataac
     1441 gacaccgaag gatttaacat tgagagcaag gatctgaagt ctgaatacaa gggccaaaac
     1501 atgcgcgtaa atacgaatgc cttccgcaat gtcgatggat cgggactggt gagcaaattg
     1561 atcggcttat gtaagaagat tatcccgcct acaaacatcc gtgagaactt atacaaccgc
     1621 actgccctcg aggatcagaa cgcgaccggc ggtgaccaaa atgccacagg tggcgatcaa
     1681 aacgccaccg gcggtgacca gaatgcgaca gtcgaccatc accatcatca ccattaaaag
     1741 cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc
     1801 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     1861 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     1921 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     1981 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     2041 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     2101 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     2161 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2221 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2281 gtgtcgcccg acgtcgatat ccggatgaag gcacgaaccc agtggacata agcctgttcg
     2341 gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc
     2401 gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt
     2461 ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt
     2521 ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta
     2581 aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc
     2641 gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg
     2701 gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt
     2761 gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga
     2821 gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg
     2881 tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt
     2941 gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca
     3001 agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct
     3061 gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac
     3121 tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta
     3181 accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc
     3241 cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc
     3301 ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc
     3361 aaggtagtcg gcaaataatg tctaacaatt cgttcaagcc gacggatatc ctcgagctga
     3421 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3601 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3661 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3721 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3781 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3841 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3901 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3961 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4021 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4081 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4141 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4201 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4261 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4321 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4381 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4441 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4501 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4561 gactcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4621 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag
     4681 atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc gaatggtgca
     4741 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     4801 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     4861 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     4921 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     4981 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     5041 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     5101 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     5161 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     5221 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     5281 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     5341 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     5401 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     5461 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     5521 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     5581 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     5641 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     5701 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     5761 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     5821 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5881 gagcgcaacg caattaatgt aagttagcgc gaattgatct ggtttgacag cttatcatcg
     5941 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg
     6001 tgcaggtcgt aaa
//
