LOCUS       sequence-369578-        6999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB coupled to GFP tagged Gab2 with Shp2 binding sites mutated to 
            Phe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6999)
  AUTHORS   Fauser J, Huyot V, Matsche J, Szynal BN, Alexeev Y, Kota P, Karginov
            AV
  TITLE     Dissecting protein tyrosine phosphatase signaling by engineered 
            chemogenetic control of its activity.
  JOURNAL   J Cell Biol. 2022 Aug 1;221(8). pii: 213352. doi: 
            10.1083/jcb.202111066. Epub 2022 Jul 13.
  PUBMED    35829702
REFERENCE   2  (bases 1 to 6999)
  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"; journalName: "J Cell 
            Biol."; date: "2022-08-1"; volume: "221(8). pii: 213352. doi"; 
            pages: " 10.1083/jcb.202111066. Epub 2022 Jul 13"
COMMENT     Sequence Label: FRB-GFP-Gab2(Y604F/Y634F)
FEATURES             Location/Qualifiers
     source          1..6999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             625..903
                     /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             925..1635
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMGELYK"
     primer_bind     complement(964..985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1225..1244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1572..1593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3787..3908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3824..3843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3878..3897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3915..4370)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4052..4073)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4264..4283
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4397..4501
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4503..4860
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4503..4694
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4526..4546)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4711..4846
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4895..5689
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4895..5689
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4949..4968)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5559..5578
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5877..5896)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5921..5968
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6297..6885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6786..6805
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggcttc tagaatcctc tggcatgaga tgtggcatga aggcctggaa
      661 gaggcatctc gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag
      721 cccttgcatg ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag
      781 gcctatggtc gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg
      841 aatgtcaagg acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca
      901 aagactagtg gtggcagtgg aaccagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      961 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1021 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1081 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1141 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1201 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1261 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1321 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1381 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1441 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1501 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1561 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1621 ggcgagctgt acaagtccgg actcagatct cgaggtatga gcggcggcgg cggcgacgac
     1681 gtggtgtgta ccggctggct gaggaaatcg cctcccgaga agaagttgag gcgctatgcc
     1741 tggaagaaac gctggtttat acttcggagt ggccgaatga gtggagatcc agatgttctg
     1801 gaatactaca agaatgagca ctccaagaaa cccctgcgga tcatcaacct gaacttgtgt
     1861 gagcaggtgg atgcaggcct gaccttcaac aagaaagagc tgcaggatag ttttgtgttt
     1921 gatatcaaga ccagcgagcg cacattttac ctggtggctg agacagaggc tgacatgaat
     1981 aagtgggtcc agagcatctg ccagatctgc ggcttcaatc aggctgaaga gagcacagac
     2041 tccctgagga acctttcttc agccagtcat ggtccccgct cttctccagc tgagttcagc
     2101 tccagtcagc acctgctccg agaacggaag tcctcagccc cttcacactc tagccagcct
     2161 actttattca cgtttgagcc ccctgtgtca agccacatgc agcctacctt gtccaccagt
     2221 gcacctcagg agtatctcta cttgcaccag tgcataagca gaaggacaga aaatgcaagg
     2281 agtgccagct tctctcaggg cacccggcag aagagtgata cagctgtgca aaaacttgcc
     2341 cagagcaatg gacactgtat caacggcgtc ggaggtcaag tccatggctt ctatagcctt
     2401 cccaagccaa gccgacacaa tacagaattc aaagacagta cttatgatct cccacggagc
     2461 ctggcttccc atggccacac caagagcagc ctcacagggt ctgagactga taacgaggat
     2521 gtgtacacct tcaagatgcc cagcaacacc ctgtgtcggg aacttggaga cctccttgtg
     2581 gacaatatgg atgtcccaac cactcctctc tcagcctacc agatccctag aacattcaca
     2641 ctggacaaga accacaatgc catgacagtg gccactcctg gagattcagc catagctccc
     2701 ccaccccggc cacccaagcc aagtcaggca gaaacatctc aatggggcag cattcagcaa
     2761 agacctccaa tcagcgaaaa tagcagatct gtagctgcta ctatccccag gcgcaatacc
     2821 ctccctgcaa tggacaacag ccgactccat cgagcttctt cctgtgagac ctacgagtac
     2881 ccggcacgag gcagtgggga aagtgccagc tggtctgctg aacctccagg aaagactgcc
     2941 gtaggtcgat caaatagtgc cagctctgat gacaactacg tgcccatgaa cccaggttct
     3001 tctaccctgc tggctatgga acgaccaggg gacaactccc agagtgtcta catccccatg
     3061 agcccaggac cccatcactt tgacccactt ggctacccgt ccacagccct tcctattcac
     3121 agaggcccca gccgaggaag tgagatccag ccacccccgg tcaaccgaaa cctcaagcct
     3181 gacagaaaag caaagccaac accccttgac ctgagaaaca acactgtcat cgatgagctg
     3241 cccttcaagt cacctgtcac caagtcttgg tccaggatca acagccacac ctttaactcc
     3301 agttcctccc agtactgccg tcctatctcc acacagagca tcaccagcac agactcggga
     3361 gacagtgagg agaactatgt ccctatgcaa aacccagtat ctgcatcccc tgttcccagt
     3421 ggcactaaca gcccagctcc aaagaagagt actggcagtg tggattttct cgccctggac
     3481 ttccagccgg gctccccaag ccctcaccgc aagccatcca catcatctgt cacatcagat
     3541 gagaaggtag actttgtcca agtggataaa gagaagaccc aggccctgca gaacaccatg
     3601 caggagtgga cagatgtgcg gcagtcctcc gaaccttcca agggtgccaa gctgtaagga
     3661 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     3721 gctttaaaaa acctccaaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3781 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3841 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3901 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     3961 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     4021 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     4081 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     4141 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     4201 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     4261 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     4321 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     4381 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4441 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4501 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4561 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4621 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4681 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4741 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4801 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4861 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4921 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4981 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     5041 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     5101 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     5161 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     5221 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     5281 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     5341 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     5401 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     5461 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     5521 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     5581 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     5641 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     5701 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5761 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5821 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     5881 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     5941 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     6001 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     6061 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     6121 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     6181 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6241 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6301 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6361 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6421 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6481 aactctgtat caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6541 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6601 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6661 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6721 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6781 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6841 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6901 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6961 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
