LOCUS       sequence-243763-        4034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyDock to bind SpyTag non-covalently for affinity 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4034)
  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 4034)
  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: SpyDock
FEATURES             Location/Qualifiers
     source          1..4034
                     /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             308..646
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDCSGKTISTWISDGHVKDFYLYPGKYTFVATAAPDGYEVATPITFTVNEDGQVTVNG
                     EATEGDAHT"
     CDS             308..646
                     /codon_start=1
                     /product="improved SpyCatcher variant that shows rapid 
                     reaction with SpyTag002 (Keeble et al., 2017)"
                     /label=SpyCatcher002
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDCSGKTISTWISDGHVKDFYLYPGKYTFVATAAPDGYEVATPITFTVNEDGQVTVNG
                     EATEGDAHT"
     CDS             726..743
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(796..814)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      810..857
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      894..1349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(981..1000)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1191..1212
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1375..1479
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1480..1548,1549..2340)
                     /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(1698..1717)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2511..3099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3000..3019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3253..3270
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3285..3424
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3350..3369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3510..3532
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3526..3717)
                     /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 cgccatggta accaccttat caggtttatc aggtgagcaa ggtccgtccg gtgatatgac
      361 aactgaagaa gatagtgcta cccatattaa attctcaaaa cgtgatgagg acggccgtga
      421 gttagctggt gcaactatgg agttgcgtga ttgctctggt aaaactatta gtacatggat
      481 ttcagatgga catgtgaagg atttctacct gtatccagga aaatatacat ttgtcgcgac
      541 cgcagcacca gacggttatg aggtagcaac tccaattacc tttacagtta atgaggacgg
      601 tcaggttact gtaaatggcg aagcaactga aggtgacgct catactggat ccagtggtag
      661 ctaaatggtt gatgcttgag gatccgaatt cgagctccgt cgacaagctt gcggccgcac
      721 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
      781 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      841 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      901 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      961 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1021 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1081 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1141 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1201 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1261 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1321 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     1381 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1441 ccctggtaaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1501 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1561 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1621 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1681 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     1741 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     1801 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     1861 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     1921 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     1981 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     2041 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2101 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2161 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2221 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2281 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2341 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2401 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2461 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2521 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2581 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2641 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2701 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2761 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2821 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2881 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2941 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3001 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3061 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3121 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3181 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3241 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3301 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3361 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3421 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3481 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3541 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3601 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agcttctcca gaagcgttaa
     3661 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3721 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3781 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     3841 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     3901 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     3961 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4021 gaaaccgaag acca
//
