LOCUS       sequence-425308-        5302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes a protein (Cys-30nmSAH-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 5302)
  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 5302)
  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-30nmSAH-SPYcatcher
FEATURES             Location/Qualifiers
     source          1..5302
                     /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             1235..1573
                     /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      1614..1708
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1756..1775)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1767..2582
                     /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             1767..2582
                     /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(1840..1859)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2801..2887
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2980..4062)
                     /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     4024..4043
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4063..4140)
                     /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    4332..4472
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4387..4406
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4487..4504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4658..5246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4738..4757)
                     /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 cgcgccaagc catggtaacc accttatcag gtttatcagg
     1261 tgagcaaggt ccgtccggtg atatgacaac tgaagaagat agtgctaccc atattaaatt
     1321 ctcaaaacgt gatgaggacg gccgtgagtt agctggtgca actatggagt tgcgtgattc
     1381 atctggtaaa actattagta catggatttc agatggacat gtgaaggatt tctacctgta
     1441 tccaggaaaa tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactcc
     1501 aattgaattt acagttaatg aggacggtca ggttactgta gatggtgaag caactgaagg
     1561 tgacgctcat actggatcca gtggtagcta gtaagcttaa ttagctgagc ttggactcct
     1621 gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg
     1681 ccgccgggcg ttttttattg gtgagaatcc aagctagcca tgaaaataaa ctgtctgctt
     1741 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta
     1801 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     1861 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     1921 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     1981 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     2041 atgatgcttg gttactcacg actgcgatcc ccggcaaaac agcattccag gtattagaag
     2101 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     2161 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     2221 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     2281 gctggcctgt tgaacaagtc tggaaagaaa tgcacaaact tttgccattc tcaccggatt
     2341 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     2401 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     2461 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     2521 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     2581 aagaattaat tcatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     2641 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     2701 caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     2761 ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca
     2821 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     2881 gacaaatccg ccctctagat tacgtgcagt cgatgataag ctgtcaaaca tgagaattgt
     2941 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3001 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3061 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     3121 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     3181 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     3241 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     3301 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     3361 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     3421 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     3481 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     3541 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     3601 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     3661 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacac gttgcgcgag
     3721 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3781 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3841 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3901 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3961 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     4021 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     4081 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc
     4141 ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag ctgcctcgcg
     4201 cgtttcggtg atgacggtga aaacctctga cacattcagc tcccggagac ggtcacagct
     4261 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4321 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     4381 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     4441 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     4501 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4561 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4621 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4681 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4741 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4801 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4861 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt atgcacgaac
     4921 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4981 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5041 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5101 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5161 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5221 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5281 ctcagtggaa cgaaaactca cg
//
