LOCUS       sequence-442166-        7585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Minimal PUFFHalo cassette with s36GFP-HaloTag and mCherry-3NLS, 
            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 7585)
  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 7585)
  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: pTL52_pyCAG-s36GFPHaloTag-IRES-mCherry3NLS-polyA
FEATURES             Location/Qualifiers
     source          1..7585
                     /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..4008
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     misc_feature    4059..4631
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4225..4242)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4452..4471
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      4640..4649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4646..5353
                     /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(4791..4809)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5059..5078
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5375..5395
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5399..5419
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5423..5443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     5553..5569
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5553..5569
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    5578..5626
                     /note="synthetic polyadenylation signal"
     terminator      5861..5890
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      5912..6499
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6400..6419
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6511..6538)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(6562..7353,7354..7422))
                     /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     7185..7204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7423..7525)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(7526..7557)
                     /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 gggggggggg 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 gcagaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct gggcgagcgc
     3181 atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct gcacggtaac
     3241 ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc
     3301 attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg ttatttcttc
     3361 gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga agaggtcgtc
     3421 ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg caatccagag
     3481 cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg ggacgaatgg
     3541 ccagaatttg cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg
     3601 atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg
     3661 actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca
     3721 ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg
     3781 gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc
     3841 accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac
     3901 tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg
     3961 atcggcagcg agatcgcgcg ctggctgtcg acgctggaga tttccggcta agcggccgcg
     4021 gtaccgctcg ataagcttga tatcgaattc cgcccccccc cctctccctc ccccccccta
     4081 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     4141 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     4201 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     4261 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     4321 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     4381 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     4441 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     4501 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     4561 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     4621 cacgatgata atcgtcgccg ccaccatggt gagcaagggc gaggaggata acatggccat
     4681 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     4741 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     4801 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     4861 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     4921 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     4981 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     5041 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     5101 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     5161 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     5221 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     5281 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     5341 cgagctgtac aagacctcag gcaggacagg tagtccaaaa aagaagagaa aggtagatcc
     5401 aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtattataag cggccgctcg
     5461 atagcaaatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     5521 cgaaaggagc gcacgacccc atgcatcgat gatctagaac tagtggatcc ccgatccaat
     5581 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgggct tgggggaggg
     5641 ggaggccaga atgactccaa gagctacagg aaggcaggtc agagacccca ctggacaaac
     5701 agtggctgga ctctgcacca taacacacaa tcaacagggg agtgagctgg atccggccct
     5761 ataaggcgcg ccgtgcgata ataacgataa cgaattcaag cttgatatca ttcaggacga
     5821 gcctcagact ccagcgtaac tggactgcaa tcaactcact ggctcacctt cacgggtggg
     5881 cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6001 taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa atactgttct
     6061 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6121 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6181 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6241 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6301 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6361 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6421 tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat gctcgtcagg
     6481 ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa ggtgagccag
     6541 gtgattacat ttgggccctc attaccaatg cttaatcagt gaggcaccta tctcagcgat
     6601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatgcg
     6661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc
     6721 tccagattta tcagcaataa accagccagc cgggagggcc gagcgcagaa gtgatcctgc
     6781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg ccagaagtaa
     7021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7081 gccatccgtg agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgta aactctcaag
     7261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc attttagctt ccttagctcc
     7441 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     7501 gttggaacct cttacgtgcc gatcaagtca aaagcctccg gtcggaggct tttgactttc
     7561 tgctatggag gtcaggtatg attta
//
