LOCUS       sequence-369577-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB coupled to GFP tagged Gab1 with Shp2 binding sites mutated to 
            Phe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  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 7086)
  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-Gab1(Y628F/Y660F)
FEATURES             Location/Qualifiers
     source          1..7086
                     /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="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     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
                     VTAAGITLGMDELYK"
     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    3874..3995
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3911..3930)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3965..3984
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4002..4457)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4139..4160)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4351..4370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4484..4588
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4590..4947
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4590..4781
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4613..4633)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4798..4933
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4982..5776
                     /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             4982..5776
                     /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(5036..5055)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5646..5665
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5964..5983)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6008..6055
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6384..6972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6873..6892
                     /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 gacgagctgt acaagtccgg actcagatct cgaggtatga gcggcggcga agtggtttgc
     1681 tcgggatggc tccgcaagtc gcccccggag aagaagttga agcgttatgc gtggaagaga
     1741 aggtggtttg tgttgcgcag tggccgtttg actggagacc cggatgtcct ggagtattac
     1801 aaaaacgatc atgccaagaa gcctattcgg attattgatt taaatttatg tcagcaagtt
     1861 gatgctgggt tgacattcaa caaaaaggag tttgaaaaca gctatatctt tgatatcaac
     1921 accatcgacc ggattttcta cttggtggca gatagtgagg aagacatgaa caagtgggtc
     1981 cgttgtatct gtgacatctg tggattcaat cccacagaag aagatcctgt gaagccgctg
     2041 actggctcct cacaagcacc cgtcgattca cctttcgcta taagtacagc accagcctcc
     2101 agtcagatgg aagcttcttc agtcgcgcta cctcctcctt accaggtcat cagccttccg
     2161 ccacacccag acaccctcgg cctccaggac gatccacaag actacctctt gctgatcaac
     2221 tgtcaaagca agaagcctga acctaacaga accctctttg actctgccaa gcccaccttt
     2281 tctgagacag actgcaatga caacgtccct tcccaccaga ctcctgcttc ctcccagagc
     2341 aaacacggaa tgaatggctt tttccagcaa caaatgatgt atgactgccc accgtcccgg
     2401 ctgacatctg tctcgggaga gtccagcctc tataacctgc ccaggagcta ttaccatgac
     2461 gtgttgccaa aggaatcccc atcaagcacg gaggccgacg gggagctgta cacctttaac
     2521 accccatctg ggactgcagg tgtagaaacg cagatgagac atgtatccat cagttacgac
     2581 attccgccaa cacctggcaa cacttaccag atcccacgga catttccaga aagcacactg
     2641 ggacagtcat caaagctgga caccattcct gatatccccc cacctcggcc accaaagcca
     2701 catccaactc atgaccggtc tcctgtggaa acgtgtggag tcccacgcac ggcctcggac
     2761 actgacagca gttactgtat ccctcctcca gcaggcatga cgccctcccg gagtaatacc
     2821 atttccaccg tggatttgaa caagttgcgg aaagatgcta gttctcaaga ttgctatgat
     2881 attccacgga cctttccgag cgatagatct agttccctgg aaggcttcca tagccagtat
     2941 aaaatcaaaa gcgtgttgac agcgggaggt gtctcgggtg aagagctgga tgagaactac
     3001 gttcccatga accccaactc gccacctcga caacattccg gcagctttac cgagccaatc
     3061 caggagccaa actatgtgcc aatgacccca gggacctttg acttttcttc ctttggaatg
     3121 caagtccctc ctcctgctca tatgggcttc aggtccagcc caaagacccc tcccaggagg
     3181 ccagttcctg ttgctgactg tgaaccaccc ccggtggata ggaacctcaa gccagacaga
     3241 aaagtcaagc cggcaccttt agacataaaa cctctgtcag aatgggaaga gctgcaagcc
     3301 ccagtcagat ctcccatcac caggagcttc gctcgggact cctctaggtt tcccatgtcc
     3361 cctcggcctg attctgtgca cagtacgaca tcgagcagcg actctcatga cagtgaagag
     3421 aactatgtcc ccatgaatcc aaatctgtct ggcgaagacc cgaatctctt tgccagcaac
     3481 agccttgatg ggggaagcag cccgatgaat aaacccaaag gagacaaaca agtcgaattc
     3541 ctggatttag acctagattc tgggaagtcc actccaccac ggaagcaaaa gagcagtggt
     3601 tctggcagca gcatggcaga cgagagggtg gatttcgttg tggtggacca acagaagact
     3661 ctggccctga agagtaccag agaagcttgg acggatggga ggcagtccac agagtccgag
     3721 acacccacca agaatgtgaa gtgaggatcc accggatcta gataactgat cataatcagc
     3781 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     3841 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     3901 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3961 agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat
     4021 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     4081 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     4141 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     4201 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     4261 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     4321 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     4381 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     4441 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4501 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4561 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     4621 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4681 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4741 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4801 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4861 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4921 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     4981 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     5041 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     5101 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     5161 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     5221 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     5281 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     5341 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     5401 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     5461 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     5521 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     5581 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5641 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5701 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5761 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5821 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5881 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5941 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     6001 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     6061 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     6121 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     6181 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     6241 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6301 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6361 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6421 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6481 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6541 tagccgtagt taggccacca cttcaagaac tctgtatcac cgcctacata cctcgctctg
     6601 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6661 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6721 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6781 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6841 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6901 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6961 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7021 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7081 atgcat
//
