LOCUS       sequence-425316-        5362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes a protein (Cys-3HB-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 5362)
  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 5362)
  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-3HB-SPYcatcher
FEATURES             Location/Qualifiers
     source          1..5362
                     /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             1295..1633
                     /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      1674..1768
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1816..1835)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1827..2642
                     /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             1827..2642
                     /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(1900..1919)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2861..2947
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3040..4122)
                     /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
                     EACKAAVHNLLAQRASGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4084..4103
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4123..4200)
                     /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."
     primer_bind     complement(4284..4306)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4392..4532
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4447..4466
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4547..4564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4718..5306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4798..4817)
                     /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 ggtaccctgt ataaacaaat
      601 ggtgcaggag ctggaaaaag cccgtgaccg catggagaag ttatacaaag aaatggtgga
      661 gttaatccag aaagcgatcg aactgatgcg taaaattttc caggaagtaa aacaggaggt
      721 tgagaaggcc attgaggaga tgaaaaaatt gtacgatgaa gctaaaaaaa agattgagca
      781 aatgattcaa caaattaagc agggaggcga taaacagaaa atggaagagt tacttaagcg
      841 tgccaaagaa gagatgaaaa aagtcaaaga caagatggaa aaattattag aaaagttaaa
      901 gcaaatcatg caggaagcca aacaaaagat ggagaagctg ttaaaacaat taaaggaaga
      961 gatgaaaaaa atgaaagaaa aaatggaaaa gttattgaag gaaatgaagc agcgcatgga
     1021 ggaagtcaaa aagaagatgg acggggatga tgaattgtta gaaaagatta aaaaaaatat
     1081 cgatgattta aagaaaattg ccgaggattt aatcaaaaaa gccgaggaaa acatcaagga
     1141 ggccaaaaag atcgcagagc agctggttaa gcgcgcaaaa caattaatcg aaaaagcaaa
     1201 gcaagtagca gaggagctta ttaagaagat cttgcaattg attgaaaaag caaaggaaat
     1261 tgctgaaaag gttctgaaag gcggcagtgc catggtaacc accttatcag gtttatcagg
     1321 tgagcaaggt ccgtccggtg atatgacaac tgaagaagat agtgctaccc atattaaatt
     1381 ctcaaaacgt gatgaggacg gccgtgagtt agctggtgca actatggagt tgcgtgattc
     1441 atctggtaaa actattagta catggatttc agatggacat gtgaaggatt tctacctgta
     1501 tccaggaaaa tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactcc
     1561 aattgaattt acagttaatg aggacggtca ggttactgta gatggtgaag caactgaagg
     1621 tgacgctcat actggatcca gtggtagcta gtaagcttaa ttagctgagc ttggactcct
     1681 gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg
     1741 ccgccgggcg ttttttattg gtgagaatcc aagctagcca tgaaaataaa ctgtctgctt
     1801 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta
     1861 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     1921 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     1981 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     2041 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     2101 atgatgcttg gttactcacg actgcgatcc ccggcaaaac agcattccag gtattagaag
     2161 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     2221 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     2281 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     2341 gctggcctgt tgaacaagtc tggaaagaaa tgcacaaact tttgccattc tcaccggatt
     2401 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     2461 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     2521 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     2581 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     2641 aagaattaat tcatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     2701 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     2761 caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     2821 ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca
     2881 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     2941 gacaaatccg ccctctagat tacgtgcagt cgatgataag ctgtcaaaca tgagaattgt
     3001 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3061 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3121 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     3181 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     3241 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     3301 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     3361 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     3421 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     3481 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     3541 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     3601 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     3661 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     3721 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactggcgc gttgcgcgag
     3781 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3841 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3901 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3961 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     4021 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     4081 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     4141 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc
     4201 ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag ctgcctcgcg
     4261 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     4321 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4381 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     4441 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     4501 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     4561 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4621 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4681 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4741 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4801 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4861 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4921 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt atgcacgaac
     4981 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5041 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5101 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5161 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5221 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5281 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5341 ctcagtggaa cgaaaactca cg
//
