LOCUS       sequence-408448-        7529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golgi membrane-targeted RasAR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7529)
  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 7529)
  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(+)-Golgi-RasAR
FEATURES             Location/Qualifiers
     source          1..7529
                     /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"
     primer_bind     complement(3120..3137)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3126..3350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3396..3824
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3483..3502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3693..3714
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3833..3853)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3838..4167
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4018..4153
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4080..4099
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4234..5028
                     /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(4288..4307)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4898..4917
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5202..5323
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5239..5258)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5293..5312
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5372..5388)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5372..5388)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5385..5407)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5396..5412
                     /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(5420..5426,5427..5444,5445..5450))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5465..5486
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5603..5620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5774..6362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5854..5873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6533..7324,7325..7393))
                     /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     7156..7175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7394..7498)
                     /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 caaaggtagt gggactgcct ccggaacgga
     2701 gccacagcag tctttcagcg aggcacaaca gcagctgtgt aacactcgac aagaggttaa
     2761 cgaactcaga aagctcctgg aagaggaacg ggatcagcga gtagccgcgg agaacgcact
     2821 ttctgtggcc gaagagcaga tccggcggct ggaacactca gagtgggata gtagcaggac
     2881 ccctatcatc ggaagttgtg gtacacaaga gcaggctctg ctgatcgact tgacctccaa
     2941 tagctgcaga aggacaagat ctggggtcgg ctggaaaagg gtgctgcggt ctctctgcca
     3001 cagtcgcacc agagttcctc tgctggcggc tatctatttc ctcatgatcc atgtgctgct
     3061 gatactttgt tttactggac acctgtaatc tagagggccc gtttaaaccc gctgatcagc
     3121 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3181 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3241 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3301 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3361 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3421 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3481 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3541 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3601 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3661 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3721 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3781 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3841 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3901 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3961 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4021 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4081 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     4141 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4201 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4261 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4321 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4381 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4441 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4501 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4561 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4621 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4681 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4741 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4801 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4861 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4921 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4981 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5041 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5101 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5161 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5221 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5281 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5341 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5401 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5461 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5521 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5581 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5641 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5701 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5761 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5821 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5881 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5941 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6001 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6061 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6121 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6181 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6241 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6301 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6361 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6421 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6481 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6541 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6601 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6661 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6721 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6781 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6841 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6901 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6961 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7021 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7081 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7141 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7201 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7261 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7321 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7381 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7501 catttccccg aaaagtgcca cctgacgtc
//
