LOCUS       sequence-251715-        5487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of GFP in S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5487)
  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 5487)
  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 GFP
FEATURES             Location/Qualifiers
     source          1..5487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     93..115
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     107..124
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     108..124
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        130..556
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     CDS             join(588..590,591..1304)
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             588..1301
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(618..646)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1241..1261
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1326..1343)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1345..1479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             1954..2553
                     /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(1954..1973)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2450..2470
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2686..2702)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2687..2703)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    2718..2852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2995..3011)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2995..3011)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3008..3030)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3019..3035
                     /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(3043..3049,3050..3067,3068..3073))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3088..3109
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3226..3243)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3397..3985)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3477..3496)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4156..4947,4948..5016))
                     /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     4779..4798
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5017..5121)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5189..5207
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5245..5267)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5367..5386
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
       61 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
      121 cagtgaatta attcgttgca ggacaggatg tggtgcccga tgtgactagc tctttgctgc
      181 aggccgtcct atcctctggt tccgataaga gacccagaac tccggccccc caccgcccac
      241 cgccaccccc atacatatgt ggtacgcaag taagagtgcc tgcgcatgcc ccatgtgccc
      301 caccaagagc tttgcatccc atacaagtcc ccaaagtgga gaaccgaacc aattcttcgc
      361 gggcagaaca aaagcttctg cacacgtctc cactcgaatt tggagccggc cggcgtgtgc
      421 aaaagaggtg aatcgaacga aagacccgtg tgtaaagccg cgtttccaaa atgtataaaa
      481 ccgagagcat ctggccaatg tgcatcagtt gtggtcagca gcaaaatcaa gtgaatcatc
      541 tcagtgcaac taaagggggg atctagatcg gggtacctac tagtcaaatg agcaaaggag
      601 aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc
      661 acaaattttc tgtccgtgga gagggtgaag gtgatgctac aaacggaaaa ctcaccctta
      721 aatttatttg cactactgga aaactacctg ttccgtggcc aacacttgtc actactctga
      781 cctatggtgt tcaatgcttt tcccgttatc cggatcacat gaaacggcat gactttttca
      841 agagtgccat gcccgaaggt tatgtacagg aacgcactat atctttcaaa gatgacggga
      901 cctacaagac gcgtgctgaa gtcaagtttg aaggtgatac ccttgttaat cgtatcgagt
      961 taaagggtat tgattttaaa gaagatggaa acattcttgg acacaaactc gagtacaact
     1021 ttaactcaca caatgtatac atcacggcag acaaacaaaa gaatggaatc aaagctaact
     1081 tcaaaattcg ccacaacgtt gaagatggtt ccgttcaact agcagaccat tatcaacaaa
     1141 atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg tcgacacaat
     1201 ctgtcctttc gaaagatccc aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa
     1261 ctgctgctgg gattacacat ggcatggatg agctctacaa ataggtttaa acgcccgctg
     1321 atcagcctcg actgtgcctt ctaagatcca gacatgataa gatacattga tgagtttgga
     1381 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     1441 gctttatttg taaccattat aagctgcaat aaacaagtta actctagagg atccggtgcg
     1501 tggtggtttc atgcttctgg gaacggcaaa tgggtttagg attgggaacc cctcatcatc
     1561 tgttggaata tactattcaa cctacaaaag taacgttaaa caacactact ttatatttga
     1621 tatgaatggc cacacctttt atgccataaa acatattgta agagaatacc actcttttta
     1681 ttccttcttt ccttcttgta cgttttttgc tgtaagtagg tcgtggtgct ggtgttgcag
     1741 ttgaaataac ttaaaatata aatcataaaa ctcaaacata aacttgacta tttatttatt
     1801 tattaagaaa ggaaatataa attataaatt acaacagatc ccccgggctg caggaattcg
     1861 atagcttgca tgcctgcagg tcggccgcca cgaccggtgc cgccaccatc ccctgaccca
     1921 cgcccctgac ccctcacaag gagacgacct tccatgaccg agtacaagcc cacggtgcgc
     1981 ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac
     2041 taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg
     2101 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     2161 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     2221 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     2281 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     2341 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     2401 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     2461 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2521 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     2581 accgaaagga gcgcacgacc ccatggctcc gaccgaagcc acccggggcg gccccgccga
     2641 ccccgcaccc gcccccgagg cccaccgact ctagatcaag cttatcgata ccgtcgacct
     2701 cgaggccagc tagcttgaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2761 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2821 aactcatcaa tgtatcttat catgtctgga tcgggtaccc aattgcattc attttatgtt
     2881 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     2941 tatggctgat tatgatcagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata
     3001 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3061 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3121 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3181 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3241 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3301 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3361 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3421 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3481 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3541 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3601 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3661 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3721 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3781 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3841 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3901 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3961 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4021 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4081 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4141 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4201 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4261 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4321 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4381 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4441 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4501 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4561 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4621 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4681 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4741 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4801 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4861 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4921 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4981 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5041 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5101 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     5161 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     5221 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     5281 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     5341 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     5401 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
     5461 gccattcagg ctgcgcaact gttggga
//
