LOCUS       sequence-409406-        7175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted RasAR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  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 7175)
  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(+)-PM-RasAR
FEATURES             Location/Qualifiers
     source          1..7175
                     /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(983..985,986..1696)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIKA
                     NFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1025..1046)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1286..1305)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1633..1654
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2021..2731
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
                     LTAAGITEGMNELYK"
     primer_bind     complement(2766..2783)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2772..2996
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3042..3470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3129..3148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3339..3360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3479..3499)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3484..3813
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3664..3799
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3726..3745
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3880..4674
                     /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(3934..3953)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4544..4563
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4848..4969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4885..4904)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4939..4958
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5018..5034)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5018..5034)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5031..5053)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5042..5058
                     /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(5066..5072,5073..5090,5091..5096))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5111..5132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5249..5266)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5420..6008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5500..5519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6179..6970,6971..7039))
                     /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     6802..6821
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7040..7144)
                     /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 ccatgggctg catcaagagc aagcgcaagg acaagggtgg
      961 aagtggtgga acaggtggta gcgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1021 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1081 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1141 gcccgtgccc tggcccaccc tcgtgaccac cctgagctgg ggcgtgcagt gcttcgcccg
     1201 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1261 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1321 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1381 cggcaacatc ctggggcaca agctggagta caacgccatc cacggcaacg tctatatcac
     1441 cgccgacaag cagaagaacg gcatcaaggc caacttcggc ctcaactgca acatcgagga
     1501 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1561 gctgctgccc gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga
     1621 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1681 ggacgagctg tacaagcgca tgcatccttc taagacaagc aacactatcc gtgttttctt
     1741 gccgaacaag caaagaacag tggtcaatgt gcgaaatgga atgagcttgc atgactgcct
     1801 tatgaaagca ctcaaggtga ggggcctgca accagagtgc tgtgcagtgt tcagacttct
     1861 ccacgaacac aaaggtaaaa aagcacgctt agattggaat actgatgctg cgtctttgat
     1921 tggagaagaa cttcaagtag atttcctggg aggtagtgga ggtacaggag gtagtcagaa
     1981 tcattttgtg gacgaatacg accccactat aggaggtaca tctaaaggtg aagaattatt
     2041 cactggtgtt gtcccaattt tggttgaatt agatggtgat gttaatggtc acaaattttc
     2101 tgtctccggt gaaggtgaag gtgatgctac gtacggtaaa ttgaccttaa aattactctg
     2161 tactactggt aaattgccag ttccatggcc aaccttagtc actactttag gttatggtgt
     2221 tcaatgtttt gctagatacc cagatcatat gaaacaacat gactttttca agtctgccat
     2281 gccagaaggt tatgttcaag aaagaactat ttttttcaaa gatgacggta actacaagac
     2341 cagagctgaa gtcaagtttg aaggtgatac cttagttaat agaatcgaat taaaaggtat
     2401 tgattttaaa gaagatggta acattttagg tcacaaattg gaatacaact ataactctca
     2461 caatgtttac atcactgctg acaaacaaaa gaatggtatc aaagctaact tcaaaattag
     2521 acacaacatt gaagatggtg gtgttcaatt agctgaccat tatcaacaaa atactccaat
     2581 tggtgatggt ccagtcttgt taccagacaa ccattactta tcctatcaat ctgccttatt
     2641 caaagatcca aacgaaaaga gagaccacat ggtcttgtta gaatttttga ctgctgctgg
     2701 tattaccgag ggtatgaatg aattgtacaa ataatctaga gggcccgttt aaacccgctg
     2761 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2821 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2881 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2941 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3001 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3061 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3121 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3181 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3241 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3301 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3361 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3421 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3481 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3541 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3661 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3721 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3781 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3841 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3901 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3961 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4021 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     4081 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4141 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4201 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4261 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4321 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4381 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4441 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4501 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4561 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4621 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4681 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4741 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4801 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4861 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4921 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4981 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5041 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5101 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5161 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5221 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5281 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5341 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5401 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5461 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5521 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5581 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5641 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5761 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5821 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5881 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5941 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6001 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6061 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6121 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6181 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6241 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6301 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6361 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6421 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6481 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6541 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6601 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6661 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6721 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6781 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6841 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6901 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6961 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7021 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7081 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7141 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
