LOCUS       sequence-243766-        4242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher002-oTet for tetramerization of SpyTag-proteins 
            via tetrameric coiled-coil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4242)
  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 4242)
  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-oTet
FEATURES             Location/Qualifiers
     source          1..4242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     192..211
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        192..210
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             242..264
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             284..301
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             323..343
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             350..688
                     /codon_start=1
                     /product="improved SpyCatcher variant that shows rapid 
                     reaction with SpyTag002 (Keeble et al., 2017)"
                     /label=SpyCatcher002
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     EATKGDAHT"
     CDS             934..951
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1004..1022)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1018..1065
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1102..1557
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1189..1208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1399..1420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1583..1687
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1688..1756,1757..2548)
                     /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(1906..1925)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2719..3307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3208..3227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3461..3478
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3493..3632
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3558..3577)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3718..3740
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3734..3925)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
       61 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
      121 acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatctcga
      181 tcccgcgaaa ttaatacgac tcactatagg gagaccacaa cggtttccct ctagaaataa
      241 ttttgtttaa ctttaagaag gagatataca tatgtcgtac taccatcacc atcaccatca
      301 cgattacgac atcccaacga ccgaaaacct gtattttcag ggcgccatgg taaccacctt
      361 atcaggttta tcaggtgagc aaggtccgtc cggtgatatg acaactgaag aagatagtgc
      421 tacccatatt aaattctcaa aacgtgatga ggacggccgt gagttagctg gtgcaactat
      481 ggagttgcgt gattcatctg gtaaaactat tagtacatgg atttcagatg gacatgtgaa
      541 ggatttctac ctgtatccag gaaaatatac atttgtcgaa accgcagcac cagacggtta
      601 tgaggtagca actgctatta cctttacagt taatgagcaa ggtcaggtta ctgtaaatgg
      661 cgaagcaact aaaggtgacg ctcatactgg atccagtggt agcggcagcg gctctagtga
      721 ctattccgat ttacagcgcg ttaaacagga attgttggag gaggtcaaga aggagttgca
      781 gaaggtcaaa gaagaaatca tcgaggcatt cgtgcaagag ctgcgtaaac gcggcagtgg
      841 ttcgggcgaa ccggaagcgt aataagcttt aaatggttga tgcttgagga tccgaattcg
      901 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
      961 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1021 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1081 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1141 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1201 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1261 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1321 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1381 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1441 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1501 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1561 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1621 aaatatgtat ccgctcatga gacaataacc ctggtaaatg cttcaataat attgaaaaag
     1681 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1741 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1801 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1861 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1921 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1981 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     2041 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     2101 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2161 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2221 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2281 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2341 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2401 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2461 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2521 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2581 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2641 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2701 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2761 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2821 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2881 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2941 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3001 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3061 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3121 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3181 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3241 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3301 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3361 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3421 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3481 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3541 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3601 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3661 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3721 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3781 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3841 gctcgttgag cttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3901 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3961 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4021 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4081 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4141 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4201 tccgcgtttc cagactttac gaaacacgga aaccgaagac ca
//
