LOCUS       sequence-454035-        4899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Wnt-responsive destablized iRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4899)
  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 4899)
  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: pHM066
FEATURES             Location/Qualifiers
     source          1..4899
                     /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             249..1196
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    1369..1490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1380..1399)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1434..1453
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   1771..1833
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1945..1964)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2064..2086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2124..2142)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2210..2314
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2315..2383,2384..3175)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVSGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2533..2552)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3346..3934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3835..3854
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4088..4105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4222..4243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4258..4263,4264..4281,4282..4288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4296..4312
                     /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     4301..4323
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4320..4336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4320..4336
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4371..4405
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4734..4782
                     /note="synthetic polyadenylation signal"
     misc_feature    4796..4887
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4836..4855
                     /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 ggctgaagga tccgtcgcca ggcagcctga cctcttgacc tgcgacgatg
      301 agccgatcca tatccccggt gccatccaac cgcatggact gctgctcgcc ctcgccgccg
      361 acatgacgat cgttgccggc agcgacaacc ttcccgaact caccggactg gcgatcggcg
      421 ccctgatcgg ccgctctgcg gccgatgtct tcgactcgga gacgcacaac cgtctgacga
      481 tcgccttggc cgagcccggg gcggccgtcg gagcaccgat cactgtcggc ttcacgatgc
      541 gaaaggacgc aggcttcatc ggctcctggc atcgccatga tcagctcatc ttcctcgagc
      601 tcgagcctcc ccagcgggac gtcgccgagc cgcaggcgtt cttccgccgc accaacagcg
      661 ccatccgccg cctgcaggcc gccgaaacct tggaaagcgc ctgcgccgcc gcggcgcaag
      721 aggtgcggaa gattaccggc ttcgatcggg tgatgatcta tcgcttcgcc tccgacttca
      781 gcggcgaagt gatcgcagag gatcggtgcg ccgaggtcga gtcaaaacta ggcctgcact
      841 atcctgcctc aaccgtgccg gcgcaggccc gtcggctcta taccatcaac ccggtacgga
      901 tcattcccga tatcaattat cggccggtgc cggtcacccc agacctcaat ccggtcaccg
      961 ggcggccgat tgatcttagc ttcgccatcc tgcgcagcgt ctcgcccgtc catctggaat
     1021 tcatgcgcaa cataggcatg cacggcacga tgtcgatctc gattttgcgc ggcgagcgac
     1081 tgtggggatt gatcgtttgc catcaccgaa cgccgtacta cgtcgatctc gatggccgcc
     1141 aagcctgcga gctagtcgcc caggttctgg cctggcagat cggcgtgatg gaagagaagc
     1201 ttagccatgg cttcccgccg gaggtggagg agcaggatga tggcacgctg cccatgtctt
     1261 gtgcccagga gagcgggatg gaccgtcacc ctgcagcctg tgcttctgct aggatcaatg
     1321 tgtgataatt ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg
     1381 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1441 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     1501 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagc
     1561 gaaaggagcg cacgacccca tgcatcgata ctagttaaaa gttttgttac tttatagaag
     1621 aaattttgag tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga
     1681 atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa
     1741 ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacgcat gattatcttt
     1801 aacgtacgtc acaatatgat tatctttcta gggttaatct agtatacgcg tatgcggccg
     1861 cttaattaat ccatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     1921 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     1981 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2041 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2101 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2161 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2221 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2281 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2341 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2401 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2461 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2521 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2581 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2641 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2701 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2761 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2821 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2881 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2941 ggaggcggat aaagtttcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3001 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3061 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3121 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3181 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3241 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3301 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3361 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3421 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3481 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3541 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3601 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3661 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3721 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3781 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3841 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3901 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3961 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4021 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4081 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4141 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     4201 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     4261 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4321 aggaaacagc tatgaccatg attacgccgg ggcgcgccat tctagattaa ccctagaaag
     4381 atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt tctaaatagc
     4441 gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg
     4501 tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt gagtcaaaat
     4561 gacgcatgat tatcttttac gtgactttta agatttaact catacgataa ttatattgtt
     4621 atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt atagatatca
     4681 actagaatgc tagcatgggc ccagatctcc gaggaggtac ttggagcggc cgcaataaaa
     4741 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat
     4801 acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg
     4861 caagtgcagg tgccagaaca tttctctatc gataggtac
//
