LOCUS       sequence-413133-        5164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding photocaged SpyCatcher (pSC)-cUnaG, nUnaG-SpyTag 
            (ST), and mCherry separated by P2A cleaving sequences for mammalian 
            expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5164)
  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 5164)
  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: pSC-cUnaG-P2A-nUnaG-ST-P2A-mCh
FEATURES             Location/Qualifiers
     source          1..5164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..342
                     /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"
     CDS             541..597
                     /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             886..924
                     /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             934..990
                     /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             991..1698
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1133..1151)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1401..1420
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1764..1885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1775..1794)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1829..1848
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2000..2046
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2076..2212
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2257..2328
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2554..3142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2634..2653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3313..4104,4105..4173))
                     /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     3936..3955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    4439..4487
                     /note="synthetic polyadenylation signal"
     enhancer        4568..4871
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4873..5075
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5026..5046
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(5159..5164,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggttgata ccttatcagg tttatcaagt gagcaaggtc agtccggtga tatgacaatt
       61 gaagaagata gtgctaccca tattaaattc tcatagcgtg atgaggacgg caaagagtta
      121 gctggtgcaa ctatggagtt gcgtgattca tctggtaaaa ctattagtac atggatttca
      181 gatggacaag tgaaagattt ctacctgtat ccaggaaaat atacatttgt cgaaaccgca
      241 gcaccagacg gttatgaggt agcaactgct attaccttta cagttaatga gcaaggtcag
      301 gttactgtaa atggcaaagc aactaaaggt gacgctcata ttggtggctc tggaggtgga
      361 tctggcggtg ttaaatccgt agtcaacttg gtcggagaaa aactcgtata tgtccaaaaa
      421 tgggatggca aagagacaac ctacgtgcgt gaaattaaag acgggaaact ggttgtgacg
      481 ctgacaatgg gcgacgtggt ggcagtgcgc tcgtaccggc gtgcaaccga aggaagcggt
      541 gccaccaatt tctccctcct gaagcaggct ggggacgtgg aggaaaaccc tggccccatg
      601 gttgaaaaat ttgttggtac gtggaaaata gcggattctc ataattttgg cgaatatctg
      661 aaggctatcg gggcgcctaa agagctgagt gatggaggcg atgccacgac tccaactctg
      721 tatatttcac agaaggacgg tgataaaatg accgttaaga tcgagaatgg cccgcccacc
      781 ttcctggata cacaggtaaa gtttaaactt ggtgaagaat ttgatgagtt cccgagcgac
      841 agacgcaaag gaggctctgg tggaggctct ggaggtggct ctggagcaca catagtaatg
      901 gtagacgcct acaagccgac gaagggaagc ggagctacta acttcagcct gctgaagcag
      961 gctggagacg tggaggagaa ccctggacct gtgagcaagg gcgaggagga taacatggcc
     1021 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     1081 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1141 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1201 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1261 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1321 gtgacccagg actcctccct ccaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1381 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1441 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1501 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     1561 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     1621 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1681 gacgagctgt acaagtaagg atccgcaggc ctctgctagc ttgactgact gagatacagc
     1741 gtaccttcag ctcacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1801 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1861 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1921 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtattgg
     1981 cccatctcta tcggtatcgt agcataaccc cttggggcct ctaaacgggt cttgaggggt
     2041 tttttgtgcc cctcgggccg gattgctatc taccggcatt ggcgcagaaa aaaatgcctg
     2101 atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc
     2161 cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt aaggtcgtca
     2221 gctatcctgc aggcgatctc tcgatttcga tcaagacatt cctttaatgg tcttttctgg
     2281 acaccactag gggtcagaag tagttcatca aactttcttc cctccctaat ctcattggtt
     2341 accttgggct atcgaaactt aattaaccag tcaagtcagc tacttggcga gatcgacttg
     2401 tctgggtttc gactacgctc agaattgcgt cagtcaagtt cgatctggtc cttgctattg
     2461 cacccgttct ccgattacga gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc
     2521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3421 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3541 cgcctccatc cagtctatca actgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3601 tagtttacgc aaggttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3721 gtgcaaaaaa gcggttagct ccttcggtcc tccaatcgtt gtcagaagta agttggccgc
     3781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3841 aagatgcttt tctgtgactg gtgaatactc aaccaagtca ttctgagaat agtgtatgcg
     3901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3961 tttaaaagtg ctcatcattg gaaagcgttc ttcggggcga aaactctcaa ggatcttacc
     4021 gctgttgaga tccagttcga tgtaacccac tcgcgcaccc aactgatctt cagcatcttt
     4081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4141 aataagggcg acacggaaat gttgaatact catgcgaaag cctcctgctg atcctgtctc
     4201 ttgatagatg tttgcaaaag ctaggactcg attatacgtt gaccgatcag actatcaata
     4261 tctaggttta aacagatcgg tacgctgccg tgaccaggct tgagtacaat atctgacgat
     4321 tggacttcct gggtcccaag ctggattccg actggctgac tgccaagcaa cgatatggat
     4381 cgtcgagact cgcactgaga ggatcgttct cggtacacca ggcgcgccta gcgttacaca
     4441 cacaaaaaac caacacacat ccatcttcga tggatagcga ttttattatc taactgctga
     4501 tcgagtgtag ccagatctag taatcaatta cggggtcatt agttcatagc ccatatatgg
     4561 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     4621 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     4681 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     4741 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     4801 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     4861 ctattaccat gctgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     4921 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     4981 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     5041 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcagat
     5101 ctttgtcgat cctaccatcc actcgacaca cccgccagcg gccgctgcca agcttgccgc
     5161 cacc
//
