LOCUS       sequence-409398-        7154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded FRET-based biosensor for monitoring cellular Ras
            GTPase activity. Targeted to cytosol..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7154)
  AUTHORS   Weeks R, Zhou X, Yuan TL, Zhang J
  TITLE     Fluorescent Biosensor for Measuring Ras Activity in Living Cells.
  JOURNAL   J Am Chem Soc. 2022 Sep 28;144(38):17432-17440. doi: 
            10.1021/jacs.2c05203. Epub 2022 Sep 19.
  PUBMED    36122391
REFERENCE   2  (bases 1 to 7154)
  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: 
            "10.1021/jacs.2c05203"; journalName: "J Am Chem Soc"; date: 
            "2022-09-28- 28"; volume: "144"; issue: "38"; pages: "17432-17440"
COMMENT     Sequence Label: pcDNA3.1(+)-RasAR-NES
FEATURES             Location/Qualifiers
     source          1..7154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(923..925,926..928,929..1639)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(968..989)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1229..1248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1576..1597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1964..2674
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
                     LTAAGITEGMNELYK"
     CDS             2684..2710
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(2745..2762)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2751..2975
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3021..3449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3108..3127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3318..3339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3458..3478)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3463..3792
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3643..3778
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3705..3724
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3859..4653
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3913..3932)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4523..4542
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4827..4948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4864..4883)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4918..4937
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4997..5013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4997..5013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5010..5032)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5021..5037
                     /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(5045..5051,5052..5069,5070..5075))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5090..5111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5228..5245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5399..5987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5479..5498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6158..6949,6950..7018))
                     /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     6781..6800
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7019..7123)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
      961 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1021 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1081 gctgcccgtg ccctggccca ccctcgtgac caccctgagc tggggcgtgc agtgcttcgc
     1141 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1201 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1261 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1321 ggacggcaac atcctggggc acaagctgga gtacaacgcc atccacggca acgtctatat
     1381 caccgccgac aagcagaaga acggcatcaa ggccaacttc ggcctcaact gcaacatcga
     1441 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1501 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     1561 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1621 catggacgag ctgtacaagc gcatgcatcc ttctaagaca agcaacacta tccgtgtttt
     1681 cttgccgaac aagcaaagaa cagtggtcaa tgtgcgaaat ggaatgagct tgcatgactg
     1741 ccttatgaaa gcactcaagg tgaggggcct gcaaccagag tgctgtgcag tgttcagact
     1801 tctccacgaa cacaaaggta aaaaagcacg cttagattgg aatactgatg ctgcgtcttt
     1861 gattggagaa gaacttcaag tagatttcct gggaggtagt ggaggtacag gaggtagtca
     1921 gaatcatttt gtggacgaat acgaccccac tataggaggt acatctaaag gtgaagaatt
     1981 attcactggt gttgtcccaa ttttggttga attagatggt gatgttaatg gtcacaaatt
     2041 ttctgtctcc ggtgaaggtg aaggtgatgc tacgtacggt aaattgacct taaaattact
     2101 ctgtactact ggtaaattgc cagttccatg gccaacctta gtcactactt taggttatgg
     2161 tgttcaatgt tttgctagat acccagatca tatgaaacaa catgactttt tcaagtctgc
     2221 catgccagaa ggttatgttc aagaaagaac tatttttttc aaagatgacg gtaactacaa
     2281 gaccagagct gaagtcaagt ttgaaggtga taccttagtt aatagaatcg aattaaaagg
     2341 tattgatttt aaagaagatg gtaacatttt aggtcacaaa ttggaataca actataactc
     2401 tcacaatgtt tacatcactg ctgacaaaca aaagaatggt atcaaagcta acttcaaaat
     2461 tagacacaac attgaagatg gtggtgttca attagctgac cattatcaac aaaatactcc
     2521 aattggtgat ggtccagtct tgttaccaga caaccattac ttatcctatc aatctgcctt
     2581 attcaaagat ccaaacgaaa agagagacca catggtcttg ttagaatttt tgactgctgc
     2641 tggtattacc gagggtatga atgaattgta caaaggaagt ggtctgcccc ccctggagcg
     2701 cctgaccctg taatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     2761 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2821 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2881 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2941 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3001 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3061 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3121 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3181 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3241 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3301 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3361 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3421 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3481 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3541 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3601 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3661 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3721 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3781 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3841 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3901 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3961 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4021 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4081 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4141 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4201 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4261 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4321 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4381 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4441 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4501 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4561 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4621 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4681 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4741 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4801 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4861 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4921 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4981 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5041 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5101 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5161 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5221 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5281 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5341 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5401 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5461 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5521 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5581 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5641 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5701 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5761 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5821 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5881 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5941 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6001 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6061 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6121 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6181 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6241 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6301 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6361 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6421 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6481 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6541 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6601 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6661 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6721 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6781 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6841 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6901 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6961 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7021 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7081 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7141 tgccacctga cgtc
//
