LOCUS       sequence-243776-        4233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher002-oTri for trimerization of SpyTag-proteins 
            via trimeric coiled-coil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4233)
  AUTHORS   Khairil Anuar INA, Banerjee A, Keeble AH, Carella A, Nikov GI, 
            Howarth M
  TITLE     Spy
  JOURNAL   Nat Commun. 2019 Apr 15;10(1):1734. doi: 10.1038/s41467-019-09678-w.
  PUBMED    30988307
REFERENCE   2  (bases 1 to 4233)
  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: "2019-04-15"; volume: "10(1):1734. doi"; pages: " 
            10.1038/s41467-019-09678-w"
COMMENT     Sequence Label: SpyCatcher002-oTri
FEATURES             Location/Qualifiers
     source          1..4233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    117..256
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(182..201)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     342..364
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(358..549)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1058..1077
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1058..1076
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1108..1130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1150..1167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1189..1209
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1216..1554
                     /codon_start=1
                     /product="improved SpyCatcher variant that shows rapid 
                     reaction with SpyTag002 (Keeble et al., 2017)"
                     /label=SpyCatcher002
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     EATKGDAHT"
     CDS             1791..1808
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1861..1879)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1875..1922
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1959..2414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2046..2065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2256..2277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2440..2544
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2545..2613,2614..3405)
                     /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(2763..2782)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3576..4164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4065..4084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
       61 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
      121 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
      181 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
      241 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      301 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      361 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
      421 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagcttctc
      481 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
      541 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
      601 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
      661 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
      721 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
      781 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
      841 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
      901 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
      961 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     1021 ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac tatagggaga
     1081 ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
     1141 tcgtactacc atcaccatca ccatcacgat tacgacatcc caacgaccga aaacctgtat
     1201 tttcagggcg ccatggtaac caccttatca ggtttatcag gtgagcaagg tccgtccggt
     1261 gatatgacaa ctgaagaaga tagtgctacc catattaaat tctcaaaacg tgatgaggac
     1321 ggccgtgagt tagctggtgc aactatggag ttgcgtgatt catctggtaa aactattagt
     1381 acatggattt cagatggaca tgtgaaggat ttctacctgt atccaggaaa atatacattt
     1441 gtcgaaaccg cagcaccaga cggttatgag gtagcaactg ctattacctt tacagttaat
     1501 gagcaaggtc aggttactgt aaatggcgaa gcaactaaag gtgacgctca tactggatcc
     1561 agtggtagcg gcagcggctc tgaaattgca gcaatcaaaa aagaaatcgc agccatcaag
     1621 caagagattg ccgcaattaa acaagagata gcggctatta agaaagaaat agctgccatt
     1681 aaacagcagg gcagtggttc gggcgaaccg gaagcgtaat aagctttaaa tggttgatgc
     1741 ttgaggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
     1801 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1861 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1921 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1981 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2041 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2101 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2161 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2221 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2281 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2341 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2401 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2461 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg gtaaatgctt
     2521 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2581 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2641 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2701 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2761 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     2821 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2881 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2941 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3001 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3061 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     3121 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3181 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3241 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3301 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3361 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3421 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3481 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3541 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3601 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3661 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3721 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3781 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3841 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3901 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3961 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4021 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4081 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4141 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4201 ttttgctggc cttttgctca catgttcttt cct
//
