LOCUS       sequence-251732-        6594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of a synthetic FRB-Draper receptor in S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6594)
  AUTHORS   Williamson AP, Vale RD
  TITLE     Spatial control of Draper receptor signaling initiates apoptotic 
            cell engulfment.
  JOURNAL   J Cell Biol. 2018 Nov 5;217(11):3977-3992. doi: 
            10.1083/jcb.201711175. Epub 2018 Aug 23.
  PUBMED    30139739
REFERENCE   2  (bases 1 to 6594)
  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"; doi: 
            "10.1083/jcb.201711175"; journalName: "J Cell Biol"; date: 
            "2018-11-5- 5"; volume: "217"; issue: "11"; pages: "3977-3992"
COMMENT     Sequence Label: pMT FRB-Draper INT-GFP
FEATURES             Location/Qualifiers
     source          1..6594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..28
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     222..244
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     236..253
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     237..253
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        259..685
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     CDS             777..1055
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1827..2540
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1827..2537
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1854..1882)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2477..2497
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2562..2579)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2581..2715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             3190..3789
                     /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(3190..3209)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3686..3706
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3922..3938)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3923..3939)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3954..4088
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4231..4247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4231..4247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4244..4266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4255..4271
                     /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(4279..4285,4286..4303,4304..4309))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4324..4345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4462..4479)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4633..5221)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4713..4732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5392..6183,6184..6252))
                     /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     6015..6034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6253..6357)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6425..6443
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6481..6503)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
       61 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
      121 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      181 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      241 aacgacggcc agtgaattaa ttcgttgcag gacaggatgt ggtgcccgat gtgactagct
      301 ctttgctgca ggccgtccta tcctctggtt ccgataagag acccagaact ccggcccccc
      361 accgcccacc gccaccccca tacatatgtg gtacgcaagt aagagtgcct gcgcatgccc
      421 catgtgcccc accaagagct ttgcatccca tacaagtccc caaagtggag aaccgaacca
      481 attcttcgcg ggcagaacaa aagcttctgc acacgtctcc actcgaattt ggagccggcc
      541 ggcgtgtgca aaagaggtga atcgaacgaa agacccgtgt gtaaagccgc gtttccaaaa
      601 tgtataaaac cgagagcatc tggccaatgt gcatcagttg tggtcagcag caaaatcaag
      661 tgaatcatct cagtgcaact aaagggggga tctagatcgg ggtacctact agtcaaatgt
      721 ctcgctccgt ggccttagct gtgctcgcgc tactctctct ttctggcctg gaggcaatcc
      781 tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa
      841 ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc
      901 cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc
      961 aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc caagcctggg
     1021 acctctatta tcatgtgttc cgacgaatct caaagggtag cggtagcggt agcggtggag
     1081 gtggaagcag cgcgagtgta gctctgacct tagtcctgat gaccctgttt gcgtgcataa
     1141 tcttcgcagt gttcatttac taccgccgac gtgtgtccaa tctaaaaacc gaaatcgccc
     1201 atgtacacta cacccatgat acgaatcccc cgagctggcc gcccaaccac aacttcgaca
     1261 atcccgtgta cggcatgcag gcggaaacgc gtctcctgcc caacaatatg cgttccaaga
     1321 tgaacaactt tgaccagagg agcacgatga gcacggacta cggcgatgac tgcaatgcca
     1381 gtggcagggt gggtagctat tcgatcaact acaaccacga cctgctaacg aagaacttga
     1441 acgcggaccg gaccaatccg attgtgtaca acgaatccct caaagaggag catgtctacg
     1501 acgagatcaa gcacaaggag ggctacaagg atcccgatga atacgatcac ctggactact
     1561 cacgacccag cacctcgcag aagccgcact accaccgcat gaacgacgcc atgctcaaca
     1621 tcaaccagga cgaggagaag cccagcaacg tgaataacat gactgtgctg ctgaacaaac
     1681 cgctgccgcc cacggaaccg gagccacaac acgagtgctt cgacaacacg aacaccaacc
     1741 tggacaacgt gtcgacggcg tcgcctagtt ccagtccgaa atttctaaag cggcgcgccg
     1801 gcggccgctc gagtctagag ggccccagca aaggagaaga acttttcact ggagttgtcc
     1861 caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc cgtggagagg
     1921 gtgaaggtga tgctacaaac ggaaaactca cccttaaatt tatttgcact actggaaaac
     1981 tacctgttcc gtggccaaca cttgtcacta ctctgaccta tggtgttcaa tgcttttccc
     2041 gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg
     2101 tacaggaacg cactatatct ttcaaagatg acgggaccta caagacgcgt gctgaagtca
     2161 agtttgaagg tgataccctt gttaatcgta tcgagttaaa gggtattgat tttaaagaag
     2221 atggaaacat tcttggacac aaactcgagt acaactttaa ctcacacaat gtatacatca
     2281 cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacgttgaag
     2341 atggttccgt tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg
     2401 tccttttacc agacaaccat tacctgtcga cacaatctgt cctttcgaaa gatcccaacg
     2461 aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca
     2521 tggatgagct ctacaaatag gtttaaacgc ccgctgatca gcctcgactg tgccttctaa
     2581 gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2641 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2701 tgcaataaac aagttaactc tagaggatcc ggtgcgtggt ggtttcatgc ttctgggaac
     2761 ggcaaatggg tttaggattg ggaacccctc atcatctgtt ggaatatact attcaaccta
     2821 caaaagtaac gttaaacaac actactttat atttgatatg aatggccaca ccttttatgc
     2881 cataaaacat attgtaagag aataccactc tttttattcc ttctttcctt cttgtacgtt
     2941 ttttgctgta agtaggtcgt ggtgctggtg ttgcagttga aataacttaa aatataaatc
     3001 ataaaactca aacataaact tgactattta tttatttatt aagaaaggaa atataaatta
     3061 taaattacaa cagatccccc gggctgcagg aattcgatag cttgcatgcc tgcaggtcgg
     3121 ccgccacgac cggtgccgcc accatcccct gacccacgcc cctgacccct cacaaggaga
     3181 cgaccttcca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     3241 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     3301 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     3361 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     3421 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     3481 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     3541 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     3601 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     3661 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     3721 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     3781 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
     3841 ggctccgacc gaagccaccc ggggcggccc cgccgacccc gcacccgccc ccgaggccca
     3901 ccgactctag atcaagctta tcgataccgt cgacctcgag gccagctagc ttgaacttgt
     3961 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4021 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4081 tctggatcgg gtacccaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     4141 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atcagtcgac
     4201 ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4261 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4321 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4381 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4441 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4501 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4561 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4621 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4681 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4741 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4801 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4861 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4921 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4981 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5041 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5101 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5161 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5221 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5281 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5341 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5401 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5461 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5521 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5581 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5641 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5701 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5761 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5821 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5881 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5941 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6001 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6061 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6121 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6181 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6241 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6301 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6361 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     6421 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6481 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6541 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggct
//
