LOCUS       sequence-409879-        6878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding EGFP fused to SpyTag (ST) and photocaged 
            SpyCatcher (pSC) localized to the cell membrane via CAAX tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6878)
  AUTHORS   Ruskowitz ER, Munoz-Robles BG, Strange AC, Butcher CH, Kurniawan S, 
            Filteau JR, DeForest CA
  TITLE     Spatiotemporal functional assembly of split protein pairs through a 
            light-activated SpyLigation.
  JOURNAL   Nat Chem. 2023 May;15(5):694-704. doi: 10.1038/s41557-023-01152-x. 
            Epub 2023 Apr 17.
  PUBMED    37069270
REFERENCE   2  (bases 1 to 6878)
  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"; doi: 
            "10.1038/s41557-023-01152-x"; journalName: "Nat Chem"; date: 
            "2023-05"; volume: "15"; issue: "5"; pages: "694-704"
COMMENT     Sequence Label: EGFP-ST-P2A-pSC-CAAX
FEATURES             Location/Qualifiers
     source          1..6878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /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(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             748..786
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             796..852
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             856..1194
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFS*RDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     primer_bind     complement(1375..1392)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1381..1605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1651..2079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1738..1757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1948..1969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2088..2108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2093..2422
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2273..2408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2335..2354
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2471..3496
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2471..3496
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    3626..3747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3663..3682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3717..3736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3796..3812)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3796..3812)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3809..3831)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3820..3836
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3844..3850,3851..3868,3869..3874))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3889..3910
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4027..4044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4198..4786)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4278..4297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4957..5748,5749..5817))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5580..5599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5818..5922)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5997..6016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6188..6567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6568..6771
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6722..6742
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6816..6835
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6816..6834
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(6873..6878,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggg
      721 ggaagtggag ggagtggggg gagcggggca cacatagtaa tggtagacgc ctacaagccg
      781 acgaagggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag
      841 aaccctggac ctatggttga taccttatca ggtttatcaa gtgagcaagg tcagtccggt
      901 gatatgacaa ttgaagaaga tagtgctacc catattaaat tctcatagcg tgatgaggac
      961 ggcaaagagt tagctggtgc aactatggag ttgcgtgatt catctggtaa aactattagt
     1021 acatggattt cagatggaca agtgaaagat ttctacctgt atccaggaaa atatacattt
     1081 gtcgaaaccg cagcaccaga cggttatgag gtagcaactg ctattacctt tacagttaat
     1141 gagcaaggtc aggttactgt aaatggcaaa gcaactaaag gtgacgctca tattggtgga
     1201 tctggaggtg gatctggagg tatgagcaaa gatggcaaaa agaagaaaaa gaagtcaaag
     1261 acaaagtgtg taattatgtg aggatccact agtccagtgt ggtggaattc tgcagatatc
     1321 cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     1381 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     1441 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     1501 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     1561 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     1621 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     1681 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1741 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1801 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1861 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1921 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1981 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2041 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     2101 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2161 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2221 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     2281 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2341 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     2401 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     2461 tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     2521 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     2581 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     2641 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     2701 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     2761 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga
     2821 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     2881 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
     2941 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     3001 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     3061 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     3121 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     3181 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     3241 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     3301 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     3361 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     3421 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     3481 gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
     3541 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3601 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3661 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3721 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     3781 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3841 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3901 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3961 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4021 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4081 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4141 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4201 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4261 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4321 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4381 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4441 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4501 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4561 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4621 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4681 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4741 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4801 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4861 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4921 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4981 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5041 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5101 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5161 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5221 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5281 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5341 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5401 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5461 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5521 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5581 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5641 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5701 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5761 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5821 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5881 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5941 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     6001 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     6061 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     6121 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     6181 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     6241 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     6301 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     6361 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     6421 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     6481 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     6541 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     6601 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     6661 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     6721 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
     6781 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
     6841 caagctggct agcgtttaaa cttaagcttg ccgccacc
//
