LOCUS       sequence-267051-        6260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-Talin head-SpyCatcher003 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6260)
  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 6260)
  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: EGFP-Talin head-SpyCatcher003
FEATURES             Location/Qualifiers
     source          1..6260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      243..252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(249..251,252..254,255..965)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(249..251,252..254,255..965)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(294..315)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(555..574)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     902..923
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1014..1023
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2328..2666
                     /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"
     polyA_signal    2684..2805
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2721..2740)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2775..2794
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2812..3267)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2949..2970)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3161..3180
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3294..3398
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3400..3757
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3400..3591
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3423..3443)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3608..3743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3792..4586
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3792..4586
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3846..3865)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4456..4475
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4774..4793)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4818..4865
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5194..5782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5683..5702
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5957..6260
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      301 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      361 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      421 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      481 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      541 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
      601 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
      661 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
      721 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
      781 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
      841 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
      901 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
      961 acaagtccgg actcagatct cgagctcaag ctagcttcga attcgcagaa gctgccacca
     1021 tggttgcgct ttcgctgaag attagcattg ggaatgtggt gaagacgatg caatttgagc
     1081 catctaccat ggtgtacgac gcctgccgca tgattcgtga gcggatccca gaggccctgg
     1141 ctggccctcc caacgacttt gggctctttc tgtcagatga tgaccccaaa aaaggcatct
     1201 ggctagaggc tgggaaagct ttggactact acatgctccg aaatggggac accatggagt
     1261 acagaaaaaa gcagagaccc ctgaagatcc ggatgttaga cggaacggtg aagactatca
     1321 tggtggatga ctccaagacc gtcacagaca tgctcatgac catttgtgcc cgaattggta
     1381 tcaccaacca tgatgagtat tcactggttc gagagctgat ggaagaaaag aaagatgagg
     1441 ggacagggac cctgagaaaa gacaagaccc tactgcgaga tgaaaagaag atggagaagc
     1501 taaagcagaa gttgcacaca gacgatgagt tgaactggct ggaccatggg cggacactga
     1561 gggaacaggg agtggaagag catgagacgt tgctgctgcg gagaaagttc ttctactcag
     1621 accagaatgt ggattcccga gaccctgtac agctgaacct tctctatgtg caggcacgag
     1681 atgacatcct gaatggctcc catcctgtct cctttgacaa ggcctgtgaa tttgcaggct
     1741 tccagtgcca gatccagttt ggacctcaca atgaacagaa gcacaaggct ggcttccttg
     1801 acctgaagga cttcctgccc aaggagtatg tgaagcagaa gggagagcgt aagatctttc
     1861 aggcacacaa gaattgtggg cagatgagtg aaattgaggc caaggtacgc tacgtgaagc
     1921 tggcccgttc cctcaagact tacggtgtct ccttcttcct agtcaaggaa aagatgaagg
     1981 ggaagaataa actggtgccc aggctgttgg gcatcactaa ggagtgtgtg atgcgtgtgg
     2041 atgagaagac caaggaggtg attcaggaat ggagcctcac caacatcaaa cgctgggctg
     2101 cctctcccaa gagctttact ctggactttg gagactatca ggatggctac tactcagtac
     2161 agacgacaga aggcgagcag atcgcacagc tcattgctgg ctacatagat atcatcctta
     2221 agaagaaaaa aagcaaggac cattttgggc tggagggaga tgaagagtct actatgttgg
     2281 aggactcagt gtcacccaaa aagtcaacag tccttcaggg cgcgatggtg accacactgt
     2341 ccggactgtc tggagagcag ggaccatccg gcgacatgac cacagaggag gattctgcca
     2401 cacacatcaa gttcagcaag agggacgagg acggaagaga gctggcagga gcaaccatgg
     2461 agctgaggga tagctccggc aagaccatca gcacatggat ctccgacggc cacgtgaagg
     2521 atttctacct gtatcccggc aagtacacct ttgtggagac agcagcacca gacggatatg
     2581 aggtggcaac ccctatcgag tttacagtga acgaggacgg acaggtgacc gtggatggag
     2641 aggcaacaga gggcgatgca cacacctaac aattgttgtt gttaacttgt ttattgcagc
     2701 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2761 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa
     2821 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2881 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2941 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3001 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3061 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3121 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3181 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3241 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3301 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3361 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     3421 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3481 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3541 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3601 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3661 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3721 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     3781 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3841 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3901 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3961 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4021 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4081 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4141 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4201 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4261 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     4321 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4381 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4441 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4501 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4561 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4621 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4681 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4741 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     4801 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     4861 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     4921 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     4981 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     5041 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5101 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5161 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5221 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5281 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5341 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5401 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5461 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5521 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5581 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5641 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5701 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5761 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5821 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5881 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     5941 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6001 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     6061 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     6121 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     6181 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     6241 tagtcatcgc tattaccatg
//
