LOCUS       sequence-243768-        4263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher002-oPent for pentamerization of SpyTag-proteins
            via pentameric coiled-coil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4263)
  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 4263)
  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-oPent
FEATURES             Location/Qualifiers
     source          1..4263
                     /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             955..972
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1025..1043)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1039..1086
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1123..1578
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1210..1229)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1420..1441
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1604..1708
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1709..1777,1778..2569)
                     /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(1927..1946)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2740..3328
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3229..3248
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3482..3499
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3514..3653
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3579..3598)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3739..3761
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3755..3946)
                     /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 gctctatgga
      721 cttagcaccc caaatgttgc gcgaattgca agagaccaac gctgccttgc aggatgtgcg
      781 cgagttgctt cgtcagcaag ttaaggaaat caccttcttg aagaatacgg tcatggagtg
      841 cgatgcgtgc ggaggcagtg gttcgggcga accggaagcg taataagctt taaatggttg
      901 atgcttgagg atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac
      961 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1021 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1081 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1141 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1201 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1261 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1321 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1381 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1441 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1501 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1561 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1621 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctggtaaat
     1681 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1741 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1801 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1861 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1921 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1981 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2041 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2101 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2161 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2221 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     2281 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2341 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2401 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2461 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2521 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2581 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     2641 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2701 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2761 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2821 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2881 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2941 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3001 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3061 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3121 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3181 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3241 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3301 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3361 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3421 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3481 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3541 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3601 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3661 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3721 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3781 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3841 ctgcctgttc atccgcgtcc agctcgttga gcttctccag aagcgttaat gtctggcttc
     3901 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3961 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4021 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4081 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4141 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4201 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4261 cca
//
