LOCUS       sequence-497341-        6102 bp    DNA     circular SYN 14-JAN-2026
DEFINITION  TopFlash reporter with puromycin. Used as a live readout of 
            downstream Wnt gene transcription..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6102)
  AUTHORS   Rosen SJ, Witteveen O, Baxter N, Lach RS, Hopkins E, Bauer M, Wilson
            MZ
  TITLE     Anti-resonance in developmental signaling regulates cell fate 
            decisions.
  JOURNAL   bioRxiv [Preprint]. 2025 May 23:2025.02.04.636331. doi: 
            10.1101/2025.02.04.636331.
  PUBMED    39990305
REFERENCE   2  (bases 1 to 6102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 14, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 May 23:2025.02.04.636331. doi: 
            10.1101/2025.02.04.636331."
COMMENT     Sequence Label: pPig_8X-TOPFLash-tdIRFP_Puro
FEATURES             Location/Qualifiers
     source          1..6102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   23..57
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        582..613
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2631..2684
                     /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"
     CDS             2685..3284
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2685..2704)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3181..3201
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     repeat_region   3525..3587
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3699..3718)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3818..3840
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3878..3896)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3964..4068
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4069..4137,4138..4929)
                     /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     complement(4287..4306)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5100..5688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5589..5608
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5842..5859
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5976..5997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6012..6017,6018..6035,6036..6042)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6050..6066
                     /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)."
     primer_bind     6055..6077
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6074..6090
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6074..6090
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggggcgcgcc attctagatt aaccctagaa agatagtctg cgtaaaattg acgcatgcat
       61 tcttgaaata ttgctctctc tttctaaata gcgcgaatcc gtcgctgtgc atttaggaca
      121 tctcagtcgc cgcttggagc tcccgtgagg cgtgcttgtc aatgcggtaa gtgtcactga
      181 ttttgaacta taacgaccgc gtgagtcaaa atgacgcatg attatctttt acgtgacttt
      241 taagatttaa ctcatacgat aattatattg ttatttcatg ttctacttac gtgataactt
      301 attatatata tattttcttg ttatagatat caactagaat gctagcatgg gcccagatct
      361 ccgaggggac ccgacaggcc cggtctgctt tttgcttgta ctgtcgaccc tgtggaatga
      421 agtgcaggtg ccagaacatt tctctatcga taggtaccga gctcttacgc gagatcaaag
      481 ggggtaagat caaagggggt aagatcaaag ggggtaagat caaagggggt aagatcaaag
      541 gggcgcgccc gcgtgctagc ccgggctcga gatctagact ctagagggta tataatggaa
      601 gctcgaattc cagcttggca ttccggtact gttggtaaaa agcttggcat tccggtactg
      661 ttggtaaagc caccatggct gaaggcagcg tggcccgaca gccagacctt ttgacttgtg
      721 acgatgaacc aatccacata ccgggggcaa tacaacctca tggtctcctt ctggcgcttg
      781 ctgccgacat gactatagtg gccggctctg acaacttgcc ggaattgacc ggacttgcta
      841 ttggggcgtt gattgggcgc tctgccgctg atgtatttga ttccgagaca cataataggc
      901 ttactatagc cctcgccgaa ccaggggctg ccgtcggcgc tcctataaca gttgggttca
      961 cgatgcgaaa agatgctggg ttcattggta gctggcatcg ccacgatcaa cttatcttcc
     1021 ttgagcttga accccctcaa caggacgttg cggaacccca agctttcttt agaaggacca
     1081 attcagccat aaggcgcctt caggccgcag agacattgga gtccgcgtgt gcggcagcag
     1141 cgcaggaagt acgaaagatc acgggatttg accgggttat gatttacaga ttcgcatctg
     1201 atttctccgg ggaagtcatc gcggaggatc ggtgtgcaga agtggaaagc aagcttggtt
     1261 tgcattaccc cgcatctacg gttccggccc aagcgaggag actgtatacg ataaacccag
     1321 tgaggatcat acctgacata aattatagac cggttcccgt tacgccagac ctgaaccccg
     1381 tcacaggcag gccaatagac ttgtcttttg caatcctgcg gtcagtctca cctgttcacc
     1441 tcgagtttat gaggaacata gggatgcatg ggacgatgag catctcaatc ctgagaggtg
     1501 aacggctctg gggacttatt gtttgtcatc atcgcacacc gtattacgtt gaccttgatg
     1561 gtcgccaggc ctgcgaactc gtagctcaag tattggcctg gcagatcggt gttatggagg
     1621 aaagcggtca tgggactggg agtacaggta gcggcagctc tagtggcacc tccatggcag
     1681 aagggtccgt agcaaggcaa cctgacttgt tgacctgtga tgatgaaccg attcacattc
     1741 ctggagcaat tcaaccgcat gggctgctcc ttgctttggc agcggacatg acgatcgtcg
     1801 ccggctccga taacctgccc gagttgacgg gcttggcgat aggagccctg ataggccgct
     1861 cagccgctga cgtattcgat agcgaaacgc ataaccggct tacaatcgcc ttggctgaac
     1921 cgggcgcggc cgtgggagca ccgattactg taggctttac aatgagaaaa gacgccggct
     1981 ttatcgggtc atggcaccga catgaccagc tgattttcct ggaattggag cccccgcagc
     2041 gggatgtagc cgaaccacag gccttcttcc ggcgcactaa ctccgcaatt aggagactgc
     2101 aggcagctga gactttggaa tcagcatgcg cggcagctgc acaagaagtc cggaaaatca
     2161 cgggttttga ccgagtcatg atctatagat tcgcgagcga tttctcagga gaagttattg
     2221 cggaagaccg atgcgcggag gtagaatcta agcttgggtt gcactacccc gcctccaccg
     2281 ttccggcgca agccagacgg ctctatacca ttaatccggt gcggatcatt ccagatataa
     2341 attaccggcc tgtacctgtg acaccggatt tgaaccctgt cacgggccga ccgatagacc
     2401 tcagcttcgc tatattgcga tctgtgtcac cggtccacct cgagtttatg aggaatatag
     2461 gcatgcatgg tacaatgtcc atttccattc tccggggtga acggctttgg ggcctcatcg
     2521 tttgtcacca tcgaacaccg tattacgtcg atctcgacgg cagacaggca tgtgagttgg
     2581 tcgctcaggt actcgcttgg cagatagggg taatggagga ggggagcgga gaaggtcgcg
     2641 gaagtttgtt gacctgcggg gacgtggaag aaaacccggg tccaatgacc gagtacaagc
     2701 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     2761 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     2821 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     2881 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     2941 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3001 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3061 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3121 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3181 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3241 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     3301 cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gatactagtt aaaagttttg
     3361 ttactttata gaagaaattt tgagtttttg ttttttttta ataaataaat aaacataaat
     3421 aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa cttaatatct
     3481 attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg tcaattttac
     3541 gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta atctagtata
     3601 cgcgtatgcg gccgcttaat taatccatgg cgcctgatgc ggtattttct ccttacgcat
     3661 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     3721 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3781 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3841 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     3901 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     3961 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4021 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4081 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4141 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4201 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4261 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4321 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4381 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4441 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4501 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4561 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4621 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4681 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4741 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     4801 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     4861 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     4921 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     4981 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5041 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5101 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5161 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5221 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5281 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5341 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5401 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5461 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5521 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5581 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5641 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5701 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5761 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5821 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     5881 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     5941 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     6001 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6061 taacaatttc acacaggaaa cagctatgac catgattacg cc
//
