LOCUS       sequence-442169-        6840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Minimal PUFFFIN cassette with s36GFP-mNeonGreen and mCherry-3NLS, 
            separated by t2A self-cleaving peptide, strong constitutive promoter
            CAG, no insulation or mammalian selection gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6840)
  AUTHORS   Lebek T, Malaguti M, Boezio GL, Zoupi L, Briscoe J, Elfick A, Lowell
            S
  TITLE     PUFFFIN: an ultra-bright, customisable, single-plasmid system for 
            labelling cell neighbourhoods.
  JOURNAL   EMBO J. 2024 Jul 12. doi: 10.1038/s44318-024-00154-w.
  PUBMED    38997504
REFERENCE   2  (bases 1 to 6840)
  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: "EMBO J. 
            2024 Jul 12. doi: 10.1038/s44318-024-00154-w."
COMMENT     Sequence Label: pTL54_pyCAG-s36GFPmNG-t2A-mCherry3NLS-polyA
FEATURES             Location/Qualifiers
     source          1..6840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    386..520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(423..442)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     477..496
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        606..909
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        911..1188
                     /label=chicken beta-actin promoter
     intron          1189..2205
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2129..2151
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2213..2232
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3121..3825
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      3373..3382
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3829..3840
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3832..3885
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      3895..3904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3901..4608
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4046..4064)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4314..4333
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4630..4650
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4654..4674
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4678..4698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     4808..4824
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4808..4824
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    4833..4881
                     /note="synthetic polyadenylation signal"
     terminator      5116..5145
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      5167..5754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5655..5674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5766..5793)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(5817..6608,6609..6677))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESLRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLATIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGSLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6440..6459
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6678..6780)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(6781..6812)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aatggtcagt attgagcgat atctagagaa ttcgtatagg agatctttaa ttaaacgcgt
       61 gctagcgtcg acgactcagc tgttgtcata gtaatgattg ggtggaaaca ttccaggcct
      121 gggtggagag gctttttgct tcctcttgca aaaccacact gacattccag gcctgggtgg
      181 agaggctttt tgcttcctct tgcaaaacca cactgccctc tggagggcag ttgcctagca
      241 actaattaaa agaggatgtc gcacggccag ctgcggtcag ttagtcactt cctgcttaac
      301 tgacttgaca ttttctattt taagagtcgg gaggaaaatt actgtgttgg aggccctccg
      361 ccatcttctg aagctgaatc gaattaactt gtttattgca gcttataatg gttacaaata
      421 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
      481 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatatcatc gtcgacgacg
      541 gacgctagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      601 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      661 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      721 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      781 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      841 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      901 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
      961 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     1021 gggggggggg ggcgcgcgcc rggggggggg ggggggggsg gggggsgggg sggggsgrgg
     1081 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     1141 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     1201 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1261 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1321 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
     1381 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
     1441 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
     1501 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
     1561 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
     1621 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
     1681 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
     1741 gccgtgccgg gcggggggtg gcggcaggkg ggggtgccgg gcggggcggg gccgccycgg
     1801 gccggggrgg gctcgggggr ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
     1861 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
     1921 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
     1981 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
     2041 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     2101 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
     2161 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg
     2221 ctggttattg tgctgtctca tcattttggc aaagaattcc tcgactccgc ctaggcgcgc
     2281 cacctcgagg ccgccaccat gaagtgggtg accttcatca gcctgctgtt tctgttcagc
     2341 agcgcctact ctagaggggg cggagcaagc aaaggcgaga ggctgtttag agggaaagtg
     2401 ccaatcctgg tggaactgaa aggagacgtg aacggccaca agttctctgt ccgaggcaag
     2461 gggaaaggag atgccacacg gggaaaactg actctgaagt ttatctgcac cacaggcaag
     2521 ctgcccgtgc cttggccaac cctggtcact accctgacat acggcgtgca gtgtttcagc
     2581 cgatatccaa aacacatgaa gcggcatgac ttctttaaaa gtgctatgcc caagggctac
     2641 gtgcaggagc gcaccatttc attcaagaag gatgggaagt acaagacccg ggccgaggtg
     2701 aagtttgaag ggagaaccct ggtcaaccgc atcaagctga aaggacgcga cttcaaggaa
     2761 aaagggaata ttctgggaca caaactgaga tacaacttca acagccataa ggtgtacatc
     2821 acagcagata agagaaaaaa cggcatcaag gccaaattca agattaggca caatgtgaag
     2881 gacggctccg tccagctggc tgatcattac cagcagaaca cacctattgg cagagggccc
     2941 gtgctgctgc ctaggaatca ttatctgagc actcgctcca aactgtctaa ggaccccaaa
     3001 gagaagcggg atcacatggt gctgctggaa tttgtcaccg ccgctggcat caaacatggg
     3061 cgggacgaga gatacaaggg cggcagcggc ggcaccagcg gcggcggcgg cggcagcggc
     3121 gtgagcaagg gcgaggagga taacatggcc tctctcccag cgacacatga gttacacatc
     3181 tttggctcca tcaacggtgt ggactttgac atggtgggtc agggcaccgg caatccaaat
     3241 gatggttatg aggagttaaa cctgaagtcc accaagggtg acctccagtt ctccccctgg
     3301 attctggtcc ctcatatcgg gtatggcttc catcagtacc tgccctaccc tgacgggatg
     3361 tcgcctttcc aggccgccat ggtagatggc tccggatacc aagtccatcg cacaatgcag
     3421 tttgaagatg gtgcctccct tactgttaac taccgctaca cctacgaggg aagccacatc
     3481 aaaggagagg cccaggtgaa ggggactggt ttccctgctg acggtcctgt gatgaccaac
     3541 tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt accccaacga caaaaccatc
     3601 atcagtacct ttaagtggag ttacaccact ggaaatggca agcgctaccg gagcactgcg
     3661 cggaccacct acacctttgc caagccaatg gcggctaact atctgaagaa ccagccgatg
     3721 tacgtgttcc gtaagacgga gctcaagcac tccaagaccg agctcaactt caaggagtgg
     3781 caaaaggcct ttaccgatgt gatgggcatg gacgagctgt acaagggtat agagggcaga
     3841 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctggcgt cgccgccacc
     3901 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     3961 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     4021 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     4081 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     4141 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     4201 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     4261 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     4321 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     4381 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     4441 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     4501 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     4561 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagac ctcaggcagg
     4621 acaggtagtc caaaaaagaa gagaaaggta gatccaaaaa agaagagaaa ggtagatcca
     4681 aaaaagaaga gaaaggtatt ataagcggcc gctcgatagc aaatgacccg caagcccggt
     4741 gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca
     4801 tcgatgatct agaactagtg gatccccgat ccaataaaag atctttattt tcattagatc
     4861 tgtgtgttgg ttttttgtgt gggcttgggg gagggggagg ccagaatgac tccaagagct
     4921 acaggaaggc aggtcagaga ccccactgga caaacagtgg ctggactctg caccataaca
     4981 cacaatcaac aggggagtga gctggatccg gccctataag gcgcgccgtg cgataataac
     5041 gataacgaat tcaagcttga tatcattcag gacgagcctc agactccagc gtaactggac
     5101 tgcaatcaac tcactggctc accttcacgg gtgggccttt cttcggtaga aaatcaaagg
     5161 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5221 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaggtaact
     5281 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5341 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5401 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5461 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5521 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5581 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5641 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5701 tgacttgagc atcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5761 agcaacgcag aaaggcccac ccgaaggtga gccaggtgat tacatttggg ccctcattac
     5821 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5881 gcctgactcc ccgtcgtgta gataactacg atgcgggagg gcttaccatc tggccccagt
     5941 gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag
     6001 ccagccggga gggccgagcg cagaagtgat cctgcaactt tatccgcctc catccagtct
     6061 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6121 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6181 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6241 agctccttcg gtcctccgat cgttgccaga agtaagttgg ccgcagtgtt atcactcatg
     6301 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtgagatg cttttctgtg
     6361 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6421 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6481 attggaaaac gttcttcggg gcgtaaactc tcaaggatct taccgctgtt gagatccagt
     6541 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6601 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6661 aaatgttgaa tactcatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     6721 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     6781 agtcaaaagc ctccggtcgg aggcttttga ctttctgcta tggaggtcag gtatgattta
//
