LOCUS       sequence-453877-        4668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Wnt-responsive destablized GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4668)
  AUTHORS   McNamara HM, Solley SC, Adamson B, Chan MM, Toettcher JE
  TITLE     Recording morphogen signals reveals mechanisms underlying gastruloid
            symmetry breaking.
  JOURNAL   Nat Cell Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9.
  PUBMED    39358450
REFERENCE   2  (bases 1 to 4668)
  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: "Nat Cell 
            Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9."
COMMENT     Sequence Label: pHM126
FEATURES             Location/Qualifiers
     source          1..4668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        156..187
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      243..252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(249..251,252..254,255..965,966..971,972..1094)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(294..315)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(555..574)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     902..923
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1138..1259
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1149..1168)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1203..1222
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   1540..1602
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1714..1733)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1833..1855
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1893..1911)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1979..2083
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2084..2152,2153..2944)
                     /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(2302..2321)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3115..3703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3604..3623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3857..3874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3991..4012
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4027..4032,4033..4050,4051..4057)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4065..4081
                     /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     4070..4092
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4089..4105
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4089..4105
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4140..4174
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4503..4551
                     /note="synthetic polyadenylation signal"
     misc_feature    4565..4656
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4605..4624
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 cgagctctta cgcgagatca aagggggtaa gatcaaaggg ggtaagatca aaggggcgcg
       61 agatcaaagg gggtaagatc aaagggggta agatcaaagg gggtaagatc aaaggggcgc
      121 gcccgcgtgc tagcccgggc tcgagatcta gactctagag ggtatataat ggaagctcga
      181 attccagctt ggcattccgg tactgttggt aaaaagcttg gcattccggt actgttggta
      241 aagccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      301 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      361 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      421 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      481 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      541 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
      601 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
      661 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
      721 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
      781 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
      841 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
      901 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
      961 acaagaagct tagccatggc ttcccgccgg aggtggagga gcaggatgat ggcacgctgc
     1021 ccatgtcttg tgcccaggag agcgggatgg accgtcaccc tgcagcctgt gcttctgcta
     1081 ggatcaatgt gtgataattc tagagtcggg gcggccggcc gcttcgagca gacatgataa
     1141 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1201 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     1261 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     1321 aaagcaagcg aaaggagcgc acgaccccat gcatcgatac tagttaaaag ttttgttact
     1381 ttatagaaga aattttgagt ttttgttttt ttttaataaa taaataaaca taaataaatt
     1441 gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca
     1501 aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat tttacgcatg
     1561 attatcttta acgtacgtca caatatgatt atctttctag ggttaatcta gtatacgcgt
     1621 atgcggccgc ttaattaatc catggcgcct gatgcggtat tttctcctta cgcatctgtg
     1681 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     1741 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1801 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1861 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     1921 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1981 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2041 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2101 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2161 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2221 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2281 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2341 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2401 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2461 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2521 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2581 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     2641 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2701 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2761 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2821 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2881 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2941 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3001 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3061 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3121 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3181 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3241 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3301 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3361 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3421 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3481 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3541 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3601 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3661 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3721 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3781 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3841 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     3901 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     3961 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     4021 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4081 atttcacaca ggaaacagct atgaccatga ttacgccggg gcgcgccatt ctagattaac
     4141 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
     4201 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
     4261 cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt tgaactataa cgaccgcgtg
     4321 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
     4381 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
     4441 tagatatcaa ctagaatgct agcatgggcc cagatctccg aggaggtact tggagcggcc
     4501 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     4561 tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
     4621 tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtac
//
