LOCUS       sequence-251768-        6664 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 6664)
  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 6664)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..6664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     193..215
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     207..224
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     208..224
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        230..656
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     CDS             691..708
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             709..744
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             826..1104
                     /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             1876..2583
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2018..2036)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2286..2305
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2603..2620)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2622..2756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             3231..3830
                     /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(3231..3250)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3727..3747
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3963..3979)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3964..3980)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3995..4129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4272..4288)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4272..4288)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4285..4307)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     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)."
     promoter        complement(join(4320..4326,4327..4344,4345..4350))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4365..4386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4503..4520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4674..5262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4754..4773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5433..6224,6225..6293))
                     /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     6056..6075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6294..6398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6466..6484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6522..6544)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6644..6663
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
       61 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      121 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      181 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaatta attcgttgca
      241 ggacaggatg tggtgcccga tgtgactagc tctttgctgc aggccgtcct atcctctggt
      301 tccgataaga gacccagaac tccggccccc caccgcccac cgccaccccc atacatatgt
      361 ggtacgcaag taagagtgcc tgcgcatgcc ccatgtgccc caccaagagc tttgcatccc
      421 atacaagtcc ccaaagtgga gaaccgaacc aattcttcgc gggcagaaca aaagcttctg
      481 cacacgtctc cactcgaatt tggagccggc cggcgtgtgc aaaagaggtg aatcgaacga
      541 aagacccgtg tgtaaagccg cgtttccaaa atgtataaaa ccgagagcat ctggccaatg
      601 tgcatcagtt gtggtcagca gcaaaatcaa gtgaatcatc tcagtgcaac taaagggggg
      661 atctagatcg gggtacctac tagtgaaatg caccatcacc atcatcacga agatcaagtt
      721 gacccaagac ttattgacgg aaaaggagga ggaggccggc ccatgatgtc tcgctccgtg
      781 gccttagctg tgctcgcgct actctctctt tctggcctgg aggcaatcct ctggcatgag
      841 atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa
      901 ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg
      961 aaggaaacat cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc
     1021 aggaagtaca tgaaatcagg gaatgtcaag gacctcctcc aagcctggga cctctattat
     1081 catgtgttcc gacgaatctc aaagggtagc ggtagcggta gcggtggagg tggaagcagc
     1141 gcgagtgtag ctctgacctt agtcctgatg accctgtttg cgtgcataat cttcgcagtg
     1201 ttcatttact accgccgacg tgtgtccaat ctaaaaaccg aaatcgccca tgtacactac
     1261 acccatgata cgaatccccc gagctggccg cccaaccaca acttcgacaa tcccgtgtac
     1321 ggcatgcagg cggaaacgcg tctcctgccc aacaatatgc gttccaagat gaacaacttt
     1381 gaccagagga gcacgatgag cacggactac ggcgatgact gcaatgccag tggcagggtg
     1441 ggtagctatt cgatcaacta caaccacgac ctgctaacga agaacttgaa cgcggaccgg
     1501 accaatccga ttgtgtacaa cgaatccctc aaagaggagc atgtctacga cgagatcaag
     1561 cacaaggagg gctacaagga tcccgatgaa tacgatcacc tggactactc acgacccagc
     1621 acctcgcaga agccgcacta ccaccgcatg aacgacgcca tgctcaacat caaccaggac
     1681 gaggagaagc ccagcaacgt gaagaacatg actgtgctgc tgaacaaacc gctgccgccc
     1741 acggaaccgg agccacaaca cgagtgcttc gacaacacga acaccaacct ggacaacgtg
     1801 tcgacggcgt cgcctagttc cagtccgaaa tttctaaagc ggcgcgccgg cggccgctcg
     1861 agtctagagg gccccgtgag caagggcgag gaggataaca tggccatcat caaggagttc
     1921 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     1981 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     2041 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     2101 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     2161 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2221 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     2281 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     2341 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     2401 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     2461 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     2521 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     2581 taagtttaaa cccgctgatc agcctcgact gtgccttcta agatccagac atgataagat
     2641 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2701 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaact
     2761 ctagaggatc cggtgcgtgg tggtttcatg cttctgggaa cggcaaatgg gtttaggatt
     2821 gggaacccct catcatctgt tggaatatac tattcaacct acaaaagtaa cgttaaacaa
     2881 cactacttta tatttgatat gaatggccac accttttatg ccataaaaca tattgtaaga
     2941 gaataccact ctttttattc cttctttcct tcttgtacgt tttttgctgt aagtaggtcg
     3001 tggtgctggt gttgcagttg aaataactta aaatataaat cataaaactc aaacataaac
     3061 ttgactattt atttatttat taagaaagga aatataaatt ataaattaca acagatcccc
     3121 cgggctgcag gaattcgata gcttgcatgc ctgcaggtcg gccgccacga ccggtgccgc
     3181 caccatcccc tgacccacgc ccctgacccc tcacaaggag acgaccttcc atgaccgagt
     3241 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg
     3301 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg
     3361 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3421 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3481 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3541 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3601 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3661 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3721 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3781 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     3841 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctccgac cgaagccacc
     3901 cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgactcta gatcaagctt
     3961 atcgataccg tcgacctcga ggccagctag cttgaacttg tttattgcag cttataatgg
     4021 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4081 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg ggtacccaat
     4141 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4201 aacctctaca aatgtggtat ggctgattat gatcagtcga cctgcaggca tgcaagcttg
     4261 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4321 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4501 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4561 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4621 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4681 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4741 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4801 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4861 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4921 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4981 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5041 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5101 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5161 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5221 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5281 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5341 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5401 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5461 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5521 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5581 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5641 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5701 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5761 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5821 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5881 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5941 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6001 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6061 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6121 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6181 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6241 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6301 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6361 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6421 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     6481 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     6541 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     6601 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     6661 gtac
//
