LOCUS       sequence-360329-        4207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destabilized EGFP fluorescent reporter for Retinoic Acid activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4207)
  AUTHORS   Rekler D, Kalcheim C
  TITLE     Completion of neural crest cell production and emigration is 
            regulated by retinoic-acid-dependent inhibition of BMP signaling.
  JOURNAL   Elife. 2022 Apr 8;11. pii: 72723. doi: 10.7554/eLife.72723.
  PUBMED    35394423
REFERENCE   2  (bases 1 to 4207)
  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: "Elife."; 
            date: "2022-04-8"; volume: "11. pii: 72723. doi"; pages: " 
            10.7554/eLife.72723"
COMMENT     Sequence Label: pGL3 RARE-d2EGFP
FEATURES             Location/Qualifiers
     source          1..4207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        79..275
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      126..261
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      292..301
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(298..300,301..303,304..1014,1015..1020,1021..1143)
                     /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(343..364)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(604..623)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     951..972
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1229..1248)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1294..1349
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1348..1367)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(1710..1726)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1710..1726)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1723..1745)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1734..1750
                     /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)."
     promoter        complement(join(1758..1764,1765..1782,1783..1788))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1803..1824
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1883..2078
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1929..2064
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2084..2218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2121..2140)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2175..2194
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2286..2303)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2457..3045)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2537..2556)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3216..4007,4008..4076))
                     /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     3839..3858
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4077..4181)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacagt tgggtcattt gaaggttagc agcccgggta gggttcaccg aaagttcact
       61 cgctcgagat ctgcgatctg catctcaatt agtcagcaac catagtcccg cccctaactc
      121 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat cgctgactaa
      181 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
      241 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tggtacctaa agccaccatg
      301 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      361 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      421 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      481 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      541 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      601 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      661 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      721 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      781 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      841 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      901 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      961 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagaagctt
     1021 agccatggct tcccgccgga ggtggaggag caggatgatg gcacgctgcc catgtcttgt
     1081 gcccaggaga gcgggatgga ccgtcaccct gcagcctgtg cttctgctag gatcaatgtg
     1141 taggcggccg cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     1201 atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca
     1261 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa
     1321 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt
     1381 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg
     1441 ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc
     1501 attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg
     1561 tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt
     1621 tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg
     1681 acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1741 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1801 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1861 aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa
     1921 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     1981 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2041 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc
     2101 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2161 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg
     2221 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2281 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2341 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2401 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2461 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2521 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2581 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2641 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2701 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2761 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2821 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2881 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2941 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3001 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3061 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3121 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3181 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3241 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3301 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3361 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3421 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3481 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3541 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3601 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3661 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3721 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3781 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3841 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3901 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3961 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4021 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4081 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4141 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4201 ccacctg
//
