LOCUS       sequence-442167-        7340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Minimal PUFFFIN cassette with s36GFP-mNeonGreen and mCherry-3NLS, 
            separated by IRES, 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 7340)
  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 7340)
  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: pTL53_pyCAG-s36GFPmNG-IRES-mCherry3NLS-polyA
FEATURES             Location/Qualifiers
     source          1..7340
                     /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..3828
                     /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"
     primer_bind     complement(4020..4037)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4247..4266
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      4395..4404
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4401..5108
                     /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(4546..4564)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4814..4833
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5130..5150
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5154..5174
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5178..5198
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     5308..5324
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5308..5324
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    5333..5381
                     /note="synthetic polyadenylation signal"
     terminator      5616..5645
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      5667..6254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6155..6174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6266..6293)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(6317..7108,7109..7177))
                     /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     6940..6959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7178..7280)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(7281..7312)
                     /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 ggggggcgct aggcgggggg gggcggggcg aggcgcgggg cggggcgagg
     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 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
     1801 gccggggagg gctcggggga 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 acaagtaagc ggccgcttga
     3841 tatcgaattc cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     3901 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     3961 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     4021 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     4081 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     4141 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     4201 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     4261 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     4321 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc
     4381 ccgaacccgt cgccgccacc atggtgagca agggcgagga ggataacatg gccatcatca
     4441 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     4501 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     4561 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     4621 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     4681 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     4741 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     4801 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     4861 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     4921 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     4981 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     5041 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     5101 tgtacaagac ctcaggcagg acaggtagtc caaaaaagaa gagaaaggta gatccaaaaa
     5161 agaagagaaa ggtagatcca aaaaagaaga gaaaggtatt ataagcggcc gctcgatagc
     5221 aaatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa
     5281 ggagcgcacg accccatgca tcgatgatct agaactagtg gatccccgat ccaataaaag
     5341 atctttattt tcattagatc tgtgtgttgg ttttttgtgt gggcttgggg gagggggagg
     5401 ccagaatgac tccaagagct acaggaaggc aggtcagaga ccccactgga caaacagtgg
     5461 ctggactctg caccataaca cacaatcaac aggggagtga gctggatccg gccctataag
     5521 gcgcgccgtg cgataataac gataacgaat tcaagcttga tatcattcag gacgagcctc
     5581 agactccagc gtaactggac tgcaatcaac tcactggctc accttcacgg gtgggccttt
     5641 cttcggtaga aaatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5701 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5761 actctttttc cgaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5821 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5881 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5941 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6001 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6061 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6121 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6181 ctgtcgggtt tcgccacctc tgacttgagc atcgattttt gtgatgctcg tcaggggggc
     6241 ggagcctatg gaaaaacgcc agcaacgcag aaaggcccac ccgaaggtga gccaggtgat
     6301 tacatttggg ccctcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6361 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atgcgggagg
     6421 gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag
     6481 atttatcagc aataaaccag ccagccggga gggccgagcg cagaagtgat cctgcaactt
     6541 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6601 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6661 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6721 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgccaga agtaagttgg
     6781 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6841 ccgtgagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6901 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6961 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgtaaactc tcaaggatct
     7021 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7081 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7141 agggaataag ggcgacacgg aaatgttgaa tactcatttt agcttcctta gctcctgaaa
     7201 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     7261 aacctcttac gtgccgatca agtcaaaagc ctccggtcgg aggcttttga ctttctgcta
     7321 tggaggtcag gtatgattta
//
