LOCUS       sequence-351724-        6854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Grb2-eDHFR(69K6) chimera fused to iRFP713.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  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 6854)
  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: pCAGGS-chGrb2/eDHFR(69K6)-iRFP713
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3719..3739)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3719..3737)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3758..3774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3876..3895)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3941..3996
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3995..4014)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4357..4373)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4381..4397
                     /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(4405..4411,4412..4429,4430..4435))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4450..4471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4530..4725
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4576..4711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4731..4865
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4768..4787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4822..4841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4933..4950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5104..5692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5184..5203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5863..6654,6655..6723))
                     /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     6486..6505
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6724..6828)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccrgggggg gggggggggg gggggggggg
      541 gggsggggsg rggsggrgag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcatagtagc
     1741 tagcagttgc tcgaccacca tggaagccat cgccaaatat gacttcaaag ctactgctga
     1801 cgatgagctg agcttcaaga ggggggacat ccttaaggtt ttgaatgaag agtgtgatca
     1861 gaactggtat aaggcagaac tcaatgggaa agatggcttc atccccaaga attacataga
     1921 aatgaaacca catccgtggc ggcccgggta caagtctggg tccgcagcca gtagcgccgg
     1981 aagtatgatc agtctgattg cggcgttagc ggtagatcgc gttatcggca tggaaaacgc
     2041 catgccgtgg aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct taaataaacc
     2101 cgtgattatg ggccgccata cctgggaatc aatcggtcgt ccgttgccag gacgcaaaaa
     2161 tattatcctc agcagtcaac cgggtacgga caaaaaaaag aaaaagaaag atcgcgtaac
     2221 gtgggtgaag tcggtggatg aagccatcgc ggcgtgtggt gacgtaccag aaatcatggt
     2281 gattggcggc ggtcgcgttt atgaacagtt cttgccaaaa gcgcaaaaac tgtatctgac
     2341 gcatatcgac gcagaagtgg aaggcgacac ccatttcccg gattacgagc cggatgactg
     2401 ggaatcggta ttcagcgaat tccacgatgc tgatgcgcag aactctcaca gctattgctt
     2461 tgagattctg gagcggcggc tcgagtcggg tggcggcgga tcttcagctg gtgctcaggt
     2521 gccacagcag ccaacggaag tccaggcact ctttgacttt gacccccagg aggatggtga
     2581 gctgggcttt cgccgaggag acttcattca tgtcatggat aactcagatc ccaattggtg
     2641 gaaaggggcc tgccatgggc agaccggcat gtttccccgc aattatgtca ccccagtgaa
     2701 ccggaacgtc ggcggccgct ctggtgctgg tagtggcgct ggttccggta tggccgaagg
     2761 gagcgtggca agacagcccg atctgctgac atgcgacgac gaaccaatcc acattccagg
     2821 agctatccag cctcatggac tgctgctggc tctggccgct gacatgacta tcgtggcagg
     2881 ctctgataac ctgccagagc tgaccgggct ggctatcgga gcactgattg gccggtccgc
     2941 agccgacgtg ttcgattctg agactcacaa tagactgacc atcgccctgg ctgaacctgg
     3001 ggctgcagtg ggagccccaa ttaccgtcgg cttcacaatg cgaaaggacg ctggctttat
     3061 cgggtcctgg caccgccatg atcagctgat ttttctggag ctggaaccac ctcagagaga
     3121 cgtggcagag ccacaggctt tctttcggag aacaaacagc gccatcaggc gcctgcaggc
     3181 cgctgagact ctggaatccg cttgcgcagc cgctgcacag gaagtgagga agatcaccgg
     3241 gttcgatcgc gtcatgatct acagattcgc ctcagacttt agcggagaag tgatcgcaga
     3301 agatcgctgt gccgaggtcg aaagtaaact gggcctgcat tacccagcct caaccgtgcc
     3361 tgcacaggca cgacggctgt atacaatcaa ccccgtgcgg atcattcccg acattaatta
     3421 cagaccagtg cccgtcacac ctgacctgaa cccagtgact gggaggccca tcgatctgag
     3481 cttcgccatt ctgcgcagcg tgtcccctgt ccacctggag tttatgagga atatcggaat
     3541 gcatggcacc atgtctatca gtattctgag aggcgaaagg ctgtgggggc tgatcgtgtg
     3601 ccaccatcgg acaccttact atgtggacct ggacggcagg caggcttgtg aactggtggc
     3661 tcaggtgctg gcttggcaga tcggcgtgat ggaagagtag gagctccagc ttttgttccc
     3721 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3781 attgttatcc gctaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt
     3841 gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat
     3901 ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc
     3961 attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca
     4021 aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg
     4081 ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct
     4141 gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt
     4201 tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc
     4261 agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga
     4321 tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4381 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4441 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4501 gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg
     4561 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4621 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4681 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta
     4741 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4801 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4861 ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4921 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4981 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5041 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5101 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5161 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5221 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5281 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5341 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5401 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5461 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5521 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5581 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5641 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5701 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5761 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5821 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5881 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5941 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6001 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6061 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6121 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6181 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6241 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6301 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6361 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6421 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6481 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6541 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6601 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6661 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6721 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6781 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6841 aaaagtgcca cctg
//
