LOCUS       sequence-361606-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AviTag and SpyTag003 linked to maltose binding protein in 
            the bacterial cytoplasm. AviTag enables site-specific biotinylation 
            by BirA. SpyTag003 enables reaction with SpyCatcher003..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  AUTHORS   Vester SK, Rahikainen R, Khairil Anuar INA, Hills RA, Tan TK, 
            Howarth M
  TITLE     SpySwitch enables pH- or heat-responsive capture and release for 
            plug-and-display nanoassembly.
  JOURNAL   Nat Commun. 2022 Jun 28;13(1):3714. doi: 10.1038/s41467-022-31193-8.
  PUBMED    35764623
REFERENCE   2  (bases 1 to 6540)
  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: "Nat 
            Commun."; date: "2022-06-28"; volume: "13(1):3714. doi"; pages: " 
            10.1038/s41467-022-31193-8"
COMMENT     Sequence Label: AviTag-SpyTag003-MBP
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     950..969
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(989..1066)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1272..1291
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1375..1394
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1375..1393
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1394..1418
                     /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             1433..1455
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1474..1491
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1501..1518
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1525..1569
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1579..1626
                     /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             1645..2742
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2716..2739
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2779..2796
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2849..2867)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2863..2910
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2947..3402
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3034..3053)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3244..3265
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3494..4309)
                     /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             complement(3494..4309)
                     /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     4217..4236
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4301..4320
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4431..5019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4920..4939
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5173..5190
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5205..5344
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5270..5289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5430..5452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5446..5637)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6446..6540,1..988))
                     /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 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
       61 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
      121 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
      181 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
      241 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
      301 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
      361 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
      421 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      481 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      541 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      601 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      661 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      721 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      781 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      841 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      901 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      961 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1021 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1081 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     1141 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     1201 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     1261 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     1321 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     1381 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     1441 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc
     1501 ctggtgccgc gcggcagcca tatgggtctg aatgatattt tcgaagcgca gaaaattgaa
     1561 tggcatgaag gtagcggacg tggcgtgcct catatcgtga tggtggacgc ctacaagcgt
     1621 tacaagggta gtggtgaaag tggtaaaatc gaagaaggta aactggtaat ctggattaac
     1681 ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga
     1741 attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
     1801 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa
     1861 tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt
     1921 acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg
     1981 ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg ggaagagatc
     2041 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa
     2101 gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
     2161 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg
     2221 accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc
     2281 gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg
     2341 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt
     2401 caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac
     2461 aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg
     2521 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg
     2581 aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat catgccgaac
     2641 atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc
     2701 ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgtaaaag
     2761 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     2821 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2881 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2941 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3001 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3061 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3121 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3181 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3241 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3301 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3361 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3421 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3481 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3541 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3601 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3661 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3721 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3781 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3841 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3901 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3961 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     4021 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     4081 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4141 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4201 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4261 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4321 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4381 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4441 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4501 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4561 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4621 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4681 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4741 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4801 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4861 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4921 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4981 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5041 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5101 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5161 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5221 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5281 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5341 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5401 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5461 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5521 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5581 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5641 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5701 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5761 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5821 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5881 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5941 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     6001 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6061 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     6121 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     6181 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     6241 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6301 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     6361 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6421 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6481 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
//
