LOCUS       sequence-425312-        5920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes a protein (Cys-60nmSAH-SPYcatcher) that is used
            for generating functionalized magnetic beads for MagIC-cryo-EM, a 
            method for single-particle cryo-EM analysis on magnetic beads..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5920)
  AUTHORS   Arimura Y, Konishi HA, Funabiki H
  TITLE     MagIC-Cryo-EM, structural determination on magnetic beads for scarce
            macromolecules in heterogeneous samples.
  JOURNAL   eLife. 2025 May 20;13:RP103486. doi: 10.7554/eLife.103486.
  PUBMED    40390365
REFERENCE   2  (bases 1 to 5920)
  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: "eLife."; 
            date: "2025-05-20"; volume: "13:RP103486. doi"; pages: " 
            10.7554/eLife.103486"
COMMENT     Sequence Label: Cys-60nmSAH-SPYcatcher
FEATURES             Location/Qualifiers
     source          1..5920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..53
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(77..91,92..97,98..114,115..121)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    97..113
                     /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)."
     protein_bind    129..145
                     /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             164..175
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             260..280
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1853..2191
                     /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"
     terminator      2232..2326
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2374..2393)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2385..3200
                     /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             2385..3200
                     /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(2458..2477)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3419..3505
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3598..4680)
                     /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     4642..4661
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4681..4758)
                     /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."
     misc_feature    4950..5090
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5005..5024
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5105..5122)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5276..5864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5356..5375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaagggatt ttggtcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
       61 tcttcacctc gagaaatcat aaaaaattta tttgctttgt gagcggataa caattataat
      121 agattcaatt gtgagcggat aacaatttca cacagaattc attaaagagg agaaattaac
      181 catgagcaag catcaccatc attcaggcca tcaccatacc ggacaccacc atcattcagg
      241 cagtcatcac cataccggcg agaacctgta ttttcagggt tcagctgcag gcggtgaaga
      301 ggataaaaag ccagccggag gggaaggtgg tggtgctcat attaacttaa aggttaaagg
      361 acaggatggc aacgaggtgt tcttccgcat taagcgcagt acgcaactga aaaaactgat
      421 gaacgcctac tgtgaccgtc aatcagttga catgactgcc attgcgttct tatttgacgg
      481 tcgccgcctg cgtgcagaac agacaccaga cgagttagaa atggaagatg gagatgagat
      541 cgatgcgatg ttgcatcaga caggcggtgc gggtacctgc ggtacccagg gcggaagcgg
      601 aacgcgtgaa gaggaagaga agaagaaaga agaggaagaa aagaaacaaa aagaagaaca
      661 agaaagactt gcaaaagaag aggcagagag aaaacaaaaa gaagaacaag aaagacttgc
      721 aaaagaagag gcagagagaa aacaaaagga ggaagaagag agaaaacaaa aggaagaaga
      781 agagagaaaa caaaaggagg aagaagaaag aaaattaaag gaggaacaag aaagaaaagc
      841 tgcagaagaa aagaaagcta aagaagaagc tgagagaaag gctaaagaag aacaagaaag
      901 gaaagctgaa gaagagagaa agaagaaaga agaggaagaa agacttgaaa gagaaagaaa
      961 agagagagaa gaacaagaaa agaaagccaa agaagaggca gagagaattg caaagttaga
     1021 ggctgaaaag aaggcagaag aagaaagaaa agccaaagaa gaagaagaga gaaaagccaa
     1081 agaagaagag gaaagaaaga agaaagagga gcaagaaaga cttgcaaaag aaaaggaaga
     1141 agcagaaaga aaagctgcag aggaaaagaa agctaaagaa gaacaagaaa gaaaagaaaa
     1201 ggaagaagca gaaagaaagg cgcgtgaaga ggaagagaag aagaaagaag aggaagaaaa
     1261 gaaacaaaaa gaagaacaag aaagacttgc aaaagaagag gcagagagaa aacaaaaaga
     1321 agaacaagaa agacttgcaa aagaagaggc agagagaaaa caaaaggagg aagaagagag
     1381 aaaacaaaag gaagaagaag agagaaaaca aaaggaggaa gaagaaagaa aattaaagga
     1441 ggaacaagaa agaaaagctg cagaagaaaa gaaagctaaa gaagaagctg agagaaaggc
     1501 taaagaagaa caagaaagga aagctgaaga agagagaaag aagaaagaag aggaagaaag
     1561 acttgaaaga gaaagaaaag agagagaaga acaagaaaag aaagccaaag aagaggcaga
     1621 gagaattgca aagttagagg ctgaaaagaa ggcagaagaa gaaagaaaag ccaaagaaga
     1681 agaagagaga aaagccaaag aagaagagga aagaaagaag aaagaggagc aagaaagact
     1741 tgcaaaagaa aaggaagaag cagaaagaaa agctgcagag gaaaagaaag ctaaagaaga
     1801 acaagaaaga aaagaaaagg aagaagcaga aagaaaggcg cgccaagcca tggtaaccac
     1861 cttatcaggt ttatcaggtg agcaaggtcc gtccggtgat atgacaactg aagaagatag
     1921 tgctacccat attaaattct caaaacgtga tgaggacggc cgtgagttag ctggtgcaac
     1981 tatggagttg cgtgattcat ctggtaaaac tattagtaca tggatttcag atggacatgt
     2041 gaaggatttc tacctgtatc caggaaaata tacatttgtc gaaaccgcag caccagacgg
     2101 ttatgaggta gcaactccaa ttgaatttac agttaatgag gacggtcagg ttactgtaga
     2161 tggtgaagca actgaaggtg acgctcatac tggatccagt ggtagctagt aagcttaatt
     2221 agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
     2281 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagccatg
     2341 aaaataaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     2401 gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     2461 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     2521 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     2581 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     2641 attttatccg tactcctgat gatgcttggt tactcacgac tgcgatcccc ggcaaaacag
     2701 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     2761 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     2821 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     2881 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cacaaacttt
     2941 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     3001 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     3061 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     3121 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     3181 tgctcgatga gtttttctaa gaattaattc atgggcaaat attatacgca aggcgacaag
     3241 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     3301 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     3361 taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca
     3421 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     3481 gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg atgataagct
     3541 gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     3601 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3661 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     3721 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     3781 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     3841 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     3901 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     3961 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4021 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4081 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4141 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4201 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4261 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4321 actgacacgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4381 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4441 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4501 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4561 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     4621 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     4681 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     4741 gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg ggtgcgcatg
     4801 atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca cattcagctc
     4861 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4921 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     4981 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     5041 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     5101 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5161 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5221 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5281 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5341 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5401 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5461 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5521 tgggctgtat gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5581 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5641 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5701 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5761 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5821 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5881 tttctacggg gtctgacgct cagtggaacg aaaactcacg
//
