LOCUS       sequence-261690-        6433 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Periplasmic expression of P. aeruginosa exotoxin A with DNNNS and 
            DQNRT internal glycosylation sites in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6433)
  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 6433)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-EPA-DNNNS-DQNRT
FEATURES             Location/Qualifiers
     source          1..6433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1054..1059,1060..1076,1077..1083)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1091..1107
                     /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     1096..1118
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             3090..3107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3149..3166)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3319..3405
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3497..3524
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3543..3634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3960..4751
                     /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      4995..5583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5484..5503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5737..5754
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5769..5909
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5835..5854)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5995..6017
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        6095..6172
                     /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             join(6173..6433,1..822)
                     /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(6192..6211)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
       61 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
      121 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
      181 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
      241 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
      301 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
      361 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
      421 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
      481 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
      541 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
      601 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
      661 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
      721 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 aagttagcgc gaattgatct ggtttgacag cttatcatcg actgcacggt gcaccaatgc
      901 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
      961 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     1021 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata
     1081 atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagaccatg ggcatgaaaa
     1141 agatttggct ggcgctggct ggtttagttt tagcgtttag cgcatcggcg tctagaggcc
     1201 ggctcggcct tgcggaagag gctttcgatt tatggaatga atgcgccaag gcgtgcgtgc
     1261 tggatctgaa agatggtgtg cgctcatccc gtatgtcagt cgatccggcg attgccgata
     1321 ctaatggcca gggcgtcctt cattactcaa tggtacttga gggaggcaac gatgccctta
     1381 agctagcaat tgataacgcc ttatccatca cctctgacgg gttaacgatt cgcctggagg
     1441 gaggagttga gcccaataaa cccgtacgct attcttacac tcgtcaagcc cgcggaagct
     1501 ggtcgttgaa ttggttagtg ccgattggcc atgagaaacc aagcaacatt aaggtgttca
     1561 tccacgagtt gaatgccggg aatcagttga gccatatgag ccctatttat actatcgaaa
     1621 tgggcgacga gctgctggcc aaattggccc gtgacgctac atttttcgtc cgtgctcacg
     1681 aatcgaatga gatgcagcct accttagcta ttagtcacgc tggcgtcagc gtagtcatgg
     1741 ctcaagccca gccccgccgc gagaaacgtt ggtcggagtg ggcttcggga aaggtcctgt
     1801 gtttacttga cccgctggac ggcgtctata attacctggc tcaacaacgt tgtaatttgg
     1861 acgacacctg ggaagggaag atttaccgcg tgctggccgg aaacccagcg aagcacgacc
     1921 ttgatattaa ggataataac aattctaccc ctacagtgat cagccatcgc ctgcattttc
     1981 ctgaaggcgg atcgttggca gcattgacgg cacatcaagc gtgtcactta ccattggaag
     2041 cattcactcg tcaccgccag cctcgtggtt gggagcagtt agaacagtgc gggtatcctg
     2101 ttcagcgctt agtcgccctg tatctggcgg cacgtttatc ctggaatcaa gttgaccagg
     2161 tcattcgcaa cgcacttgct tcaccaggtt ctggtggtga tctgggtgaa gcgattcgcg
     2221 aacaacctga acaagcgcgc ttagcattga ctttggctgc ggctgaatcg gagcgtttcg
     2281 tccgtcaggg aacgggcaac gatgaggccg gtgctgccag tgcggacgta gtatctttga
     2341 cgtgccccgt ggctaaagac cagaaccgca ccaaaggtga atgtgccgga ccagctgatt
     2401 ccggcgacgc cctgcttgaa cgcaattatc ctacgggcgc ggaatttctt ggtgacggcg
     2461 gagacgtgag tttttccacg cgtggaactc agaactggac ggtcgaacgt ttgcttcaag
     2521 ctcatcgcca attggaggaa cgcgggtacg tctttgtagg ataccacggg accttcttag
     2581 aagccgcaca gtcgattgtt ttcggtggcg ttcgcgcccg ttcacaggac ttagacgcaa
     2641 tctggcgtgg cttttacatt gccggggacc ctgcattggc atacggttat gctcaggacc
     2701 aggagccaga cgcgcgcggg cgtattcgca acggagcgtt attgcgcgtc tacgtgccac
     2761 gttggtcctt gccaggattc taccgcaccg ggcttacatt agccgcgcca gaagctgctg
     2821 gggaagtaga gcgtcttatc ggtcacccac ttcccttgcg tttggatgca attaccggtc
     2881 cagaagagga aggcggtcgt gtaacaatct taggttggcc tttggcagaa cgcacagtag
     2941 tgattccatc tgcgattccg acagaccctc gcaatgtagg tggcgacttg gacccttcgt
     3001 cgatcccaga taaagaacag gccatttccg ctttaccgga ttatgcctca cagccgggca
     3061 aaccaccccg cgaagactta aaaggtgggc accatcatca ccatcattaa aagcttggct
     3121 gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg
     3181 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
     3241 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
     3301 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     3361 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     3421 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     3481 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
     3541 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3601 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3661 ccgacgtcga tatccggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta
     3721 agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca
     3781 gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac
     3841 agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt
     3901 tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatca
     3961 tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg
     4021 agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg
     4081 gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa
     4141 caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg
     4201 agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt
     4261 atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta
     4321 tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac
     4381 atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg
     4441 atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg
     4501 gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca
     4561 aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc
     4621 agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct
     4681 cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag
     4741 tcggcaaata atgtctaaca attcgttcaa gccgacggat atcctcgagc tgagataggt
     4801 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4861 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4921 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4981 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5041 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5101 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     5161 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5221 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5281 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5341 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5401 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5461 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5521 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5581 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5641 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5701 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5761 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5821 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     5881 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgactcgc
     5941 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     6001 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt
     6061 cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc atcgaatggt gcaaaacctt
     6121 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     6181 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     6241 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     6301 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     6361 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     6421 taaatctcgc gcc
//
