LOCUS       sequence-351725-        7383 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EGFP-eDHFR(69K6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7383)
  AUTHORS   Suzuki S, Nakamura A, Hatano Y, Yoshikawa M, Yoshii T, Sawada S, 
            Atsuta-Tsunoda K, Aoki K, Tsukiji S
  TITLE     A chemogenetic platform for controlling plasma membrane signaling 
            and synthetic signal oscillation.
  JOURNAL   Cell Chem Biol. 2022 Sep 15;29(9):1446-1464.e10. doi: 
            10.1016/j.chembiol.2022.06.005. Epub 2022 Jul 13.
  PUBMED    35835118
REFERENCE   2  (bases 1 to 7383)
  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: 
            "10.1016/j.chembiol.2022.06.005"; journalName: "Cell Chem Biol"; 
            date: "2022-09-15- 15"; volume: "29"; issue: "9"; pages: 
            "1446-1464.e10"
COMMENT     Sequence Label: pPBpuro-EGFP-eDHFR(69K6)
FEATURES             Location/Qualifiers
     source          1..7383
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     309..331
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     393..412
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      453..462
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(459..461,462..464,465..1175)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(459..461,462..464,465..1175)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(504..525)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(765..784)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1112..1133
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1777..2326
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1921..1938)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2148..2167
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2329..2928
                     /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(2329..2348)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2825..2845
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2955..2972)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2961..3185
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3232..3248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3256..3272
                     /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(3280..3286,3287..3304,3305..3310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3325..3346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3580..3642
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3683..3703)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3683..3701)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3722..3738)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3746..3762
                     /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(3770..3776,3777..3794,3795..3800))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3815..3836
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3953..3970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4124..4712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4204..4223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4883..5674,5675..5743))
                     /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     5506..5525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5744..5848)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5874..6329)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6011..6032)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6223..6242
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6456..6478
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6470..6487
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6471..6487
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6497..6516
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6497..6515
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   6545..6579
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        6882..7261
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg
       61 ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga
      121 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg
      181 cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
      241 tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg
      301 cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt
      361 tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca
      421 tcattttggc aaagaattgg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc
      481 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
      541 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
      601 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
      661 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
      721 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
      781 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
      841 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
      901 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
      961 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1021 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     1081 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1141 ccgggatcac tctcggcatg gacgagctgt acaagtctgg gtccgcagcc agtagcgccg
     1201 gaagtatgat cagtctgatt gcggcgttag cggtagatcg cgttatcggc atggaaaacg
     1261 ccatgccgtg gaacctgcct gccgatctcg cctggtttaa acgcaacacc ttaaataaac
     1321 ccgtgattat gggccgccat acctgggaat caatcggtcg tccgttgcca ggacgcaaaa
     1381 atattatcct cagcagtcaa ccgggtacgg acaaaaaaaa gaaaaagaaa gatcgcgtaa
     1441 cgtgggtgaa gtcggtggat gaagccatcg cggcgtgtgg tgacgtacca gaaatcatgg
     1501 tgattggcgg cggtcgcgtt tatgaacagt tcttgccaaa agcgcaaaaa ctgtatctga
     1561 cgcatatcga cgcagaagtg gaaggcgaca cccatttccc ggattacgag ccggatgact
     1621 gggaatcggt attcagcgaa ttccacgatg ctgatgcgca gaactctcac agctattgct
     1681 ttgagattct ggagcggcgg taacccggga taagtcaact aacttaagct agcaacggtt
     1741 tccctctagc gggatcaatt ccgccccccc cccctaacgt tactggccga agccgcttgg
     1801 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
     1861 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
     1921 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
     1981 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
     2041 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
     2101 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
     2161 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
     2221 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     2281 ccccgaacca cggggacgtg gttttccttt gaaaaacacg ataataccat gaccgagtac
     2341 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     2401 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     2461 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     2521 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     2581 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     2641 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     2701 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     2761 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     2821 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     2881 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgatc gatgatctag
     2941 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3001 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3061 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3121 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3181 tatggcttct gaggcggaaa gaacctgcag cccaagcttg gcgtaatcat ggtcatagct
     3241 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3301 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3361 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atcttaaaag ttttgttact
     3421 ttatagaaga aattttgagt ttttgttttt ttttaataaa taaataaaca taaataaatt
     3481 gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca
     3541 aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat tttacgcatg
     3601 attatcttta acgtacgtca caatatgatt atctttctag ggttaatcta gtatacgcgt
     3661 ttaattaatc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     3721 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3781 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3841 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3901 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3961 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4021 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4081 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4141 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4201 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4261 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4321 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4381 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4441 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4501 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4561 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4621 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4681 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4741 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4801 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4861 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4921 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4981 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5041 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5101 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5161 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5221 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5281 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5341 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5401 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5461 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5521 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5581 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5641 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5701 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5761 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5821 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5881 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5941 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     6001 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     6061 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     6121 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     6181 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     6241 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     6301 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     6361 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     6421 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     6481 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttggggcgcg ccattctaga
     6541 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
     6601 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
     6661 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
     6721 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
     6781 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
     6841 tgttatagat atcaactaga atgctagcat gggcccatct cgacattgat tattgactag
     6901 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     6961 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     7021 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     7081 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     7141 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     7201 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     7261 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc
     7321 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg
     7381 ggg
//
