LOCUS       sequence-221300-        6210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher-mi3 in bacterial cells as platform for 
            plug-and-display vaccination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6210)
  AUTHORS   Bruun TUJ, Andersson AC, Draper SJ, Howarth M
  TITLE     Engineering a Rugged Nanoscaffold to Enhance Plug-and-Display 
            Vaccination.
  JOURNAL   ACS Nano. 2018 Jul 20. doi: 10.1021/acsnano.8b02805.
  PUBMED    30028591
REFERENCE   2  (bases 1 to 6210)
  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: "ACS Nano. 
            2018 Jul 20. doi: 10.1021/acsnano.8b02805."
COMMENT     Sequence Label: pET28a-SpyCatcher-mi3
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1283..1300
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1353..1371)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1367..1414
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1451..1906
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1538..1557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1748..1769
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1998..2813)
                     /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             complement(1998..2813)
                     /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     2721..2740
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2805..2824
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2935..3523
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3424..3443
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3677..3694
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3709..3848
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3774..3793)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3934..3956
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3950..4141)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4950..6032)
                     /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     5994..6013
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6033..6110)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 ttcaagtgat agtgctaccc atattaaatt ctcaaaacgt gatgaggacg gcaaagagtt
      361 agctggtgca actatggagt tgcgtgattc atctggtaaa actattagta catggatttc
      421 agatggacaa gtgaaagatt tctacctgta tccaggaaaa tatacatttg tcgaaaccgc
      481 agcaccagac ggttatgagg tagcaactgc tattaccttt acagttaatg agcaaggtca
      541 ggttactgta aacggcaaag caactaaagg tgacgctcat attgggggct cagggggatc
      601 tggcggctct ggcggttcaa tgaaaatgga agagcttttc aagaaacata agattgtcgc
      661 agtattgcgc gcaaacagcg tggaggaagc gaagaaaaag gctttagcag tgttcttggg
      721 aggggtgcat cttatagaga tcacgttcac agtacctgat gcggatactg ttattaagga
      781 attgtccttc ttaaaggaaa tgggggccat tattggtgca gggacagtca cttccgttga
      841 gcaagccaga aaagctgtgg aaagcggagc tgaatttata gtctccccgc acttggacga
      901 ggagatcagt caatttgcca aagaaaaagg ggtattttat atgccaggtg taatgacacc
      961 taccgagtta gtgaaagcaa tgaagctggg tcacactata ttaaagttgt ttccggggga
     1021 agttgtaggg ccacaattcg ttaaggctat gaaagggcca ttcccaaatg ttaaattcgt
     1081 tcctactggt ggtgtaaatc ttgacaatgt ctgtgagtgg tttaaagccg gtgtccttgc
     1141 cgtaggagtc ggatcggcac tggttaaggg aacgcccgtg gaggtagcgg agaaggctaa
     1201 ggctttcgtg gaaaaaattc gtggctgtac agaaggttct ggcgagcctg aggcttaata
     1261 aaagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     1321 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     1381 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     1441 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1501 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1561 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1621 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1681 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1741 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1801 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1861 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1921 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1981 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2041 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2101 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2161 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2221 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2281 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2341 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2401 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2461 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     2521 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2581 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2641 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     2701 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2761 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     2821 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     2881 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2941 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3001 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3061 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3121 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3181 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3241 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3301 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3361 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3421 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3481 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3541 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3601 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3661 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3721 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3781 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3841 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3901 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3961 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4021 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4081 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4141 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4201 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4261 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4321 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4381 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4441 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4501 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4561 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4621 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4681 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4741 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4801 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4861 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4921 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4981 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5041 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5101 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5161 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5221 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5281 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5341 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5401 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5461 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5521 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5581 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5641 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5701 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5761 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5821 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5881 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5941 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6001 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6061 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6121 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6181 gagcaccgcc gccgcaagga atggtgcatg
//
