LOCUS       sequence-243764-        4230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher002-oDi for dimerization of SpyTag-proteins via 
            dimeric coiled-coil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4230)
  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 4230)
  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-oDi
FEATURES             Location/Qualifiers
     source          1..4230
                     /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             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(992..1010)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1006..1053
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1090..1545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1177..1196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1387..1408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1571..1675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1676..1744,1745..2536)
                     /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(1894..1913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2707..3295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3196..3215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3449..3466
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3481..3620
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3546..3565)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3706..3728
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3722..3913)
                     /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 gctctgaaat
      721 tgcagcactg aaaaaagaaa tcgctgccct gaaacaagaa aatgccgcat taaaacaaga
      781 gatcgcagcg ctgaagaaag agattgcagc cttaaaacag ggcagtggtt cgggcgaacc
      841 ggaagcgtaa taagctttaa atggttgatg cttgaggatc cgaattcgag ctccgtcgac
      901 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
      961 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1021 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1081 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1141 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1201 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1261 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1321 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1381 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1441 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1501 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1561 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1621 gctcatgaga caataaccct ggtaaatgct tcaataatat tgaaaaagga agagtatgag
     1681 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1741 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1801 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1861 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1921 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1981 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2041 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2101 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2161 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2221 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2281 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2341 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2401 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2461 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2521 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2581 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2641 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2701 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2761 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2821 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2881 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2941 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3001 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3061 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3121 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3181 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3241 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3301 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3361 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3421 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3481 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3541 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3601 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3661 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3721 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3781 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagct
     3841 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3901 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     3961 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4021 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4081 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4141 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4201 gactttacga aacacggaaa ccgaagacca
//
