LOCUS       sequence-316166-        6273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher003-mi3 in bacterial cells as virus-like 
            particle platform for plug-and-display vaccination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  AUTHORS   Rahikainen R, Rijal P, Tan TK, Wu HJ, Andersson AC, Barrett JR, 
            Bowden TA, Draper SJ, Townsend AR, Howarth M
  TITLE     Overcoming Symmetry Mismatch in Vaccine Nanoassembly through 
            Spontaneous Amidation.
  JOURNAL   Angew Chem Int Ed Engl. 2020 Sep 4. doi: 10.1002/anie.202009663.
  PUBMED    32886840
REFERENCE   2  (bases 1 to 6273)
  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: "Angew Chem 
            Int Ed Engl. 2020 Sep 4. doi: 10.1002/anie.202009663."
COMMENT     Sequence Label: SpyCatcher003-mi3
FEATURES             Location/Qualifiers
     source          1..6273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(857..1195)
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     RBS             1214..1236
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1251..1275
                     /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     complement(1275..1294)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1276..1294)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1378..1397)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1603..1680
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1681..2763
                     /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(1700..1719)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3572..3763
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3757..3779)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3865..4004
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3920..3939
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4019..4036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4190..4778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4270..4289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4889..4908)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4900..5715
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4900..5715
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4973..4992)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5807..6262)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5944..5965)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6156..6175
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt
      181 attaagcctc aggctcgcca gaaccttctg tacagccacg aattttttcc acgaaagcct
      241 tagccttctc cgctacctcc acgggcgttc ccttaaccag tgccgatccg actcctacgg
      301 caaggacacc ggctttaaac cactcacaga cattgtcaag atttacacca ccagtaggaa
      361 cgaatttaac atttgggaat ggccctttca tagccttaac gaattgtggc cctacaactt
      421 cccccggaaa caactttaat atagtgtgac ccagcttcat tgctttcact aactcggtag
      481 gtgtcattac acctggcata taaaataccc ctttttcttt ggcaaattga ctgatctcct
      541 cgtccaagtg cggggagact ataaattcag ctccgctttc cacagctttt ctggcttgct
      601 caacggaagt gactgtccct gcaccaataa tggcccccat ttcctttaag aaggacaatt
      661 ccttaataac agtatccgca tcaggtactg tgaacgtgat ctctataaga tgcacccctc
      721 ccaagaacac tgctaaagcc tttttcttcg cttcctccac gctgtttgcg cgcaatactg
      781 cgacaatctt atgtttcttg aaaagctctt ccattttcat tgaaccgcca gagccgccag
      841 atccccctga gcccccagta tgagcgtcac cttcagttgc ttcaccatct acagtaacct
      901 gaccgtcctc attaactgta aattcaattg gagttgctac ctcataaccg tctggtgctg
      961 cggtttcgac aaatgtatat tttcctggat acaggtagaa atccttcaca tgtccatctg
     1021 aaatccatgt actaatagtt ttaccagatg aatcacgcaa ctccatagtt gcaccagcta
     1081 actcacggcc gtcctcatca cgttttgaga atttaatatg ggtagcacta tcttcttcag
     1141 ttgtcatatc accggacgga ccttgttcac ctgataaacc tgataaggtg gttacacttg
     1201 aacccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     1261 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     1321 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     1381 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     1441 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     1501 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     1561 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     1621 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     1681 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     1741 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     1801 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     1861 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     1921 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     1981 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     2041 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     2101 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     2161 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     2221 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     2281 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     2341 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     2401 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     2461 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     2521 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     2581 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     2641 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2761 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     2821 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     2881 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     2941 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     3001 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     3061 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     3121 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     3181 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     3241 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     3301 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     3361 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     3421 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     3481 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     3541 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     3601 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     3661 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     3721 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3781 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3841 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3901 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     3961 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     4021 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4081 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4141 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4201 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4261 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4321 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4381 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4441 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4501 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4561 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4621 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4681 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4741 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4801 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     4861 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     4921 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     4981 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     5041 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     5101 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5161 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     5221 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5281 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5341 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5401 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5461 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     5521 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     5581 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     5641 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     5701 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     5761 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5821 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5881 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5941 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6001 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6061 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6121 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6181 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6241 aatgcgccgc tacagggcgc gtcccattcg cca
//
