LOCUS       sequence-316165-        5943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Borrelia burgdorferi OspC (20-209) C129S with N-terminal 
            SpyTag003 fusion in bacterial cells .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5943)
  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 5943)
  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: SpyTag003-OspC
FEATURES             Location/Qualifiers
     source          1..5943
                     /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(773..820)
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     CDS             complement(821..838)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(848..865)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             884..906
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    921..945
                     /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(945..964)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(946..964)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1048..1067)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1273..1350
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1351..2433
                     /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(1370..1389)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3242..3433
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3427..3449)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3535..3674
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3590..3609
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3689..3706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3860..4448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3940..3959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4559..4578)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4570..5385
                     /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             4570..5385
                     /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(4643..4662)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5477..5932)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5614..5635)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5826..5845
                     /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 attacggttt tttcgggctt tctgcaacaa tcggactggt cagttcttta actgcatttt
      241 tcagggtatc ctgtgctgct ttgctcagat tttcaacggc tttaaacagt ttttccagtt
      301 cggctgcgcc tttatcttta ttggcatgtt ttttcagaat ggcacgctgg gcattatcat
      361 ctgtcagatt atctaaaccc agcacggcat gttcgctttt cagtttattg gtaaattcgg
      421 tgctgctctg ttttgcggta tcgattttct ctttcagttc ttcgtttttc agcacattca
      481 gtttttctgc aatcagatcg ctaatggcat atgcaccgct cagcaggctg gtatttttgc
      541 tctggtttgc ttccaggcca ttattaccga tttttttccc aattgctttg gttgccagtt
      601 catcaatgct tgccagcagg gtttcaactt ctttaacggc cagaacaact gcattgcttt
      661 cggtgatttt tttgctgatt tcggtcagat tcggaccttt aacgctttca tctgcgctat
      721 ttgcgcttgc attaccatct ttgccgctgt tattaccact ttcaccacta cccttgtaac
      781 gcttgtaggc gtccaccatc acgatatgag gcacgccacg gctgccgcgc ggcaccaggc
      841 cgctgctgtg atgatgatga tgatggctgc tgcccatggt atatctcctt cttaaagtta
      901 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
      961 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     1021 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     1081 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     1141 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     1201 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     1261 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     1321 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     1381 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     1441 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     1501 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     1561 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     1621 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     1681 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     1741 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     1801 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     1861 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     1921 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     1981 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     2041 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     2101 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     2161 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     2221 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     2281 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     2341 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     2401 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     2461 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     2521 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     2581 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     2641 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     2701 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     2761 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     2821 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     2881 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     2941 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     3001 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     3061 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     3121 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     3181 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     3241 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     3301 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     3361 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     3421 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     3481 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     3541 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     3601 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     3661 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3721 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3781 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3841 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3901 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3961 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4021 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4081 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4141 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4201 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4261 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4321 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4381 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4441 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4501 cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca
     4561 aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc
     4621 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     4681 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     4741 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     4801 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     4861 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     4921 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     4981 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     5041 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     5101 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     5161 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     5221 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     5281 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     5341 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag
     5401 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5461 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     5521 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5581 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5641 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5701 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5761 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5821 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5881 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     5941 cca
//
