LOCUS       sequence-267048-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher003 for display at the cell surface of mammalian
            cells, with superfolder GFP for visualization and myc tag for 
            antibody detection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6172)
  AUTHORS   Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, 
            Hytonen VP, Howarth M
  TITLE     Approaching infinite affinity through engineering of peptide-protein
            interaction.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116.
  PUBMED    31822621
REFERENCE   2  (bases 1 to 6172)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116."
COMMENT     Sequence Label: TfR-sfGFP-myc tag-SpyCatcher003
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          774..1600
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             1918..2631
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2632..2661
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2689..3027
                     /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
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     promoter        complement(3092..3110)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3093..3110)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3130..3147)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3136..3360
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3422..3521)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3633..3652)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3644..4453
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3711..3730)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4543..5131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5032..5051
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5296..5382
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5474..5501
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     5509..5532
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    5551..5650
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccgcggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta
      781 ccgcctatag agtctatagg cccaccccct tggcttctta tgcatgctat actgtttttg
      841 gcttggggtc tatacacccc cgcttcctca tgttataggt gatggtatag cttagcctat
      901 aggtgtgggt tattgaccat tattgaccac tcccctattg gtgacgatac tttccattac
      961 taatccataa catggctctt tgccacaact ctctttattg gctatatgcc aatacactgt
     1021 ccttcagaga ctgacacgga ctctgtattt ttacaggatg gggtctcatt tattatttac
     1081 aaattcacat atacaacacc accgtcccca gtgcccgcag tttttattaa acataacgtg
     1141 ggatctccac gcgaatctcg ggtacgtgtt ccggacatgg gctcttctcc ggtagcggcg
     1201 gagcttctac atccgagccc tgctcccatg cctccagcga ctcatggtcg ctcggcagct
     1261 ccttgctcct aacagtggag gccagactta ggcacagcac gatgcccacc accaccagtg
     1321 tgccgcacaa ggccgtggcg gtagggtatg tgtctgaaaa tgagctcggg gagcgggctt
     1381 gcaccgctga cgcatttgga agacttaagg cagcggcaga agaagatgca ggcagctgag
     1441 ttgttgtgtt ctgataagag tcagaggtaa ctcccgttgc ggtgctgtta acggtggagg
     1501 gcagtgtagt ctgagcagta ctcgttgctg ccgcgcgcgc caccagacat aatagctgac
     1561 agactaacag actgttcctt tccatgggtc ttttctgcag tcaccgtcct tgacacgaag
     1621 cttggtaccg ccaccatgat ggatcaagct agatcagcat tctctaactt gtttggtgga
     1681 gaaccattgt catgtacccg ggccagcctg gctcggcaag tagatggcga taacagtcat
     1741 gtggagatga aacttgctgt agatgaagaa gaaaatgctg acaataacac aaaggccaat
     1801 gtcacaaaac caaaaaggtg tagtggaagt atctgctatg ggactattgc tgtgatcgtc
     1861 tttttcttga ttggatttat gattggctac ttgggctatg gaagcggagg atccgggatg
     1921 cgtaaaggcg aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat
     1981 gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa
     2041 ctgacgctga agttcatctg tactactggt aaactgccgg taccttggcc gactctggta
     2101 acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat
     2161 gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag
     2221 gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac
     2281 cgcattgagc tgaaaggcat tgactttaaa gaagacggca atatcctggg ccataagctg
     2341 gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt
     2401 aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac
     2461 taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg
     2521 agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg
     2581 gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactatacaa agaacaaaaa
     2641 ctcatctcag aagaggatct gggttccggt ggctcgggtg gatccatggt gaccacactg
     2701 tccggactgt ctggagagca gggaccatcc ggcgacatga ccacagagga ggattctgcc
     2761 acacacatca agttcagcaa gagggacgag gacggaagag agctggcagg agcaaccatg
     2821 gagctgaggg atagctccgg caagaccatc agcacatgga tctccgacgg ccacgtgaag
     2881 gatttctacc tgtatcccgg caagtacacc tttgtggaga cagcagcacc agacggatat
     2941 gaggtggcaa cccctatcga gtttacagtg aacgaggacg gacaggtgac cgtggatgga
     3001 gaggcaacag agggcgatgc acacaccgga tctagcggat cttaagaatt cctcgaggcg
     3061 gccgctcgag catgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc
     3121 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3181 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3241 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3301 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3361 cttctgaggc ggaaagaacc agctggggct cgagggggga tcgatccgtc gagatatcta
     3421 gacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc
     3481 aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc tgcagctctg
     3541 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     3601 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     3661 ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     3721 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga
     3781 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     3841 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     3901 ccgtactcct ggtgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca
     3961 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     4021 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     4081 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     4141 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     4201 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     4261 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     4321 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     4381 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     4441 tgagtttttc taatcagaat tggttaattg gttgtaacat tattcagatt gggccccgtt
     4501 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4561 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4621 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4681 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4741 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4801 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4861 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4921 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4981 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5041 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5101 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5161 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5221 ggataaccgt attaccgcta gcatggatct cggggacgtc taactactaa gcgagagtag
     5281 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcgg aagactgggc ctttcgtttt
     5341 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     5401 aacgttgtga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     5461 catcaaacta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     5521 cctgttagtt agttacttaa gctcgggccc caaataatga ttttattttg actgatagtg
     5581 acctgttcgt tgcaacaaat tgataagcaa tgctttttta taatgccaac tttgtacaaa
     5641 aaagcaggct ccaccatggg aacccgcgtt ttgagatttc tgtcgccgac taaattcatg
     5701 tcgcgcgata gtggtgttta tcgccgatag agatggcgat attggaaaaa tcgatatttg
     5761 aaaatatggc atattgaaaa tgtcgccgat gtgagtttct gtgtaactga tatcgccatt
     5821 tttccaaaag tgatttttgg gcatacgcga tatctggcga tagcgcttat atcgtttacg
     5881 ggggatggcg atagacgact ttggtgactt gggcgattct gtgtgtcgca aatatcgcag
     5941 tttcgatata ggtgacagac gatatgaggc tatatcgccg atagaggcga catcaagctg
     6001 gcacatggcc aatgcatatc gatctataca ttgaatcaat attggccatt agccatatta
     6061 ttcattggtt atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcca
     6121 tatcataata tgtacattta tattggctca tgtccaacat taccgccatg tt
//
