LOCUS       sequence-313210-        6456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Streptococcus pyogenes Spy0469 (24-389) in bacterial 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6456)
  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 6456)
  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: Spy0469
FEATURES             Location/Qualifiers
     source          1..6456
                     /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(1337..1357)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1367..1384)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1398..1420
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1435..1459
                     /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(1459..1478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1460..1478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1562..1581)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1787..1864
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1865..2947
                     /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(1884..1903)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3755..3946
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3940..3962)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4048..4187
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4103..4122
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4202..4219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4373..4961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4453..4472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5072..5091)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5083..5898
                     /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             5083..5898
                     /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(5156..5175)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5990..6445)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6127..6148)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6339..6358
                     /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 ggtagtgctc gagtgcggcc gcaagctttt
      181 attatttcgg cggtatcggt tcattggtga tataatgttt accgttttat tacgcattga
      241 acgagacatg gacgtggtca taatgattct ctgtaatcga cccgcggtct ggcattggat
      301 tccacgtata cgccggaccg tagatggacg cgaagggtgc gtagaaacgc tgtttccaaa
      361 tgacgtagga gatgccacgt tcggccatat ggtcaatcgc gtactgcgcg acctggtctc
      421 ccagcgccga attttccggt accataaaat cgatggctaa tcccttgcca tggtctccgg
      481 ggtctcctgg gcggtaaccg gagaaactgg taatcccaaa ggcggacgct acttcttcct
      541 tgaaggccgc agtctgtggt tgcagacccg cgttcatagg gttgtaagca tggttcggag
      601 cgtaactcag accattcgat gtcgcgacag aggttgtgtc agccacagcc ttttgggccg
      661 ccggtgccga agtctcttct ttttctgctg gagcctgcgc cggtgctgcc gaagaaacct
      721 cctcgcttgc tgactcgttt gggactggac gattagctga cgtgggggct tccgaaatat
      781 ctgtcttagg ttccacctct gtcgtttctg cggctttggg agttggcaca gcttcttggg
      841 gcacctcggg ttgcttctgc gattctgagc ttaactcggt gctaacatcg ttagtagaag
      901 aggtaagctc acttgatgcg gtgacacttg aatcaggctt ggccgacgcg aaaggtgtgg
      961 ttggtacatc ggacggagta gcgggcggcg ccatagggac tgtaggctgg cttgtcgcag
     1021 acgcttgcgg caacggttcc gaagacggga catgacttac tgaggcaggt gaactggctg
     1081 gggcctgaac agtcaggttt gtcgcctgac cgtgctggtt gtagttcgcc gtcaagatag
     1141 tatccggaaa gataaggtcg atattagcaa tgtgattaat atctcccaat acgtgaacgt
     1201 cgattcccat cgcttccgca atagtgctta atgtatcacc gtactttact gtataggtaa
     1261 acacattgtc cactaatact aattccgatt taatttctgt tacgctgcgg ggtgtccatt
     1321 cttgagcctg tcctgagcct tggaagtaaa ggttttctcc gcttccatgg tggtgatgat
     1381 gatggcccat atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt
     1441 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg
     1501 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     1561 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     1621 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     1681 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     1741 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg
     1801 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     1861 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     1921 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     1981 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     2041 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     2101 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     2161 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     2221 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     2281 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     2341 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     2401 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     2461 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     2521 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     2581 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     2641 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     2701 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     2761 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     2821 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     2881 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     2941 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     3001 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     3061 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     3121 cgctctgggt catttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     3181 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     3241 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat
     3301 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca
     3361 gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac
     3421 ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa
     3481 gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     3541 tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg
     3601 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     3661 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     3721 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     3781 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     3841 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     3901 ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3961 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4021 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     4081 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc
     4141 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     4201 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4261 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4321 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4381 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4441 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4501 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4561 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4621 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4681 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4741 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4801 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4861 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4921 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4981 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca
     5041 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     5101 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     5161 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     5221 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     5281 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     5341 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     5401 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     5461 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     5521 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     5581 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     5641 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     5701 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     5761 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     5821 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     5881 ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa
     5941 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     6001 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     6061 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     6121 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     6181 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     6241 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     6301 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     6361 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     6421 cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
