LOCUS       sequence-243774-        4290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher002-oHept for heptamerization of SpyTag-proteins
            via heptameric coiled-coil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4290)
  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 4290)
  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-oHept
FEATURES             Location/Qualifiers
     source          1..4290
                     /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             982..999
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1052..1070)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1066..1113
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1150..1605
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1237..1256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1447..1468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1631..1735
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1736..1804,1805..2596)
                     /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(1954..1973)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2767..3355
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3256..3275
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3509..3526
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3541..3680
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3606..3625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3766..3788
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3782..3973)
                     /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 gctctaaaaa
      721 acaaggtgat gcagatgttt gtggcgaagt cgcctatatc caatccgttg ttagtgattg
      781 tcacgtacca actgccgaat tgcgtaccct tttggagatc cgtaagctgt ttcttgagat
      841 tcaaaaactg aaagttgaat tacagggtct tagcaaggaa ggcagtggtt cgggcgaacc
      901 ggaagcgtaa taagctttaa atggttgatg cttgaggatc cgaattcgag ctccgtcgac
      961 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1021 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1081 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1141 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1201 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1261 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1321 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1381 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1441 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1501 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1561 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1621 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1681 gctcatgaga caataaccct ggtaaatgct tcaataatat tgaaaaagga agagtatgag
     1741 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1801 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1861 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1921 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1981 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2041 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2101 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2161 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2221 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2281 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2341 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2401 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2461 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2521 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2581 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2641 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2701 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2761 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2821 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2881 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2941 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3001 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3061 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3121 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3181 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3241 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3301 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3361 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3421 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3481 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3541 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3601 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3661 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3721 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3781 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3841 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagct
     3901 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3961 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4021 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4081 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4141 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4201 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4261 gactttacga aacacggaaa ccgaagacca
//
