LOCUS       sequence-423115-        5848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes a protein (SPYtag-Histag-Avidin) 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 5848)
  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 5848)
  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: SPYtag-Histag-Avidin
FEATURES             Location/Qualifiers
     source          1..5848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     613..632
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(652..729)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     934..953
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1037..1056
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1037..1055
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1056..1080
                     /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             1095..1117
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1128..1175
                     /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             1176..1193
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1617..1634
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1687..1705)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1701..1748
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1785..2240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1872..1891)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2082..2103
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2266..2370
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2371..2439,2440..3231)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2589..2608)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3402..3990
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3891..3910
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4144..4161
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4176..4315
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4241..4260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4401..4423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4417..4608)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5417..5848,1..651))
                     /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"
ORIGIN
        1 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
       61 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      121 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      181 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      241 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      301 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      361 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      421 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      481 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      541 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      601 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
      661 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
      721 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
      781 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
      841 cgcccaacag tccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
      901 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
      961 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     1021 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     1081 cctctagaaa taattttgtt taactttaag aaggagatat acatatgcgt ggtgttccgc
     1141 acatcgtaat ggttgacgcg tacaaacgtt acaaacatca ccatcaccac catggtggtg
     1201 gcggttctgc tgaagctggt atcaccggca cctggtacaa ccagctggga tccaccttca
     1261 tcgttaccgc tggtgctgac ggtgctctga ccggtaccta cgaatccgct gttggtaacg
     1321 ctgaatctag atacgttctg accggtcgtt acgactccgc tccggctacc gacggttccg
     1381 gaaccgctct gggttggacc gttgcttgga aaaacaacta ccgtaacgct cactccgcta
     1441 ccacctggtc tggccagtac gttggtggtg ctgaagctcg tatcaacacc cagtggttgt
     1501 tgacctccgg caccaccgaa gccaacgcgt ggaaatccac cctggttggt cacgacacct
     1561 tcaccaaagt taaaccgtcc gctgcttcct aataaaagct tgcggccgca ctcgagcacc
     1621 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1681 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1741 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1801 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg acagctacac ttgccagcgc
     1861 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1921 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1981 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2041 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2101 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2161 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2221 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2281 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2341 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2401 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2461 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2521 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2581 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     2641 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2701 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2761 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2821 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2881 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     2941 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3001 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3061 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3121 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3181 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3241 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3301 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3361 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3421 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3481 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3541 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3601 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3661 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3721 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3781 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3841 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3901 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3961 tgctcgtaag gggggcggag cctatggaaa aacgccagca acgcggcctt ttcacggttc
     4021 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4081 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4141 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4201 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     4261 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4321 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4381 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4441 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4501 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4561 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccat
     4621 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4681 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4741 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4801 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4861 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4921 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4981 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5041 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     5101 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     5161 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     5221 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     5281 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     5341 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5401 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5461 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5521 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5581 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5641 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     5701 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5761 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5821 gaaaaccgga catggcactc cagtcgcc
//
