LOCUS       sequence-428815-        6478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ras activity dependent proximity labeler, Key only, plasma membrane 
            localized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6478)
  AUTHORS   Zhang JZ, Nguyen WH, Greenwood N, Rose JC, Ong SE, Maly DJ, Baker D
  TITLE     Computationally designed sensors detect endogenous Ras activity and 
            signaling effectors at subcellular resolution.
  JOURNAL   Nat Biotechnol. 2024 Jan 25. doi: 10.1038/s41587-023-02107-w.
  PUBMED    38273065
REFERENCE   2  (bases 1 to 6478)
  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: "Nat 
            Biotechnol. 2024 Jan 25. doi: 10.1038/s41587-023-02107-w."
COMMENT     Sequence Label: pcDNA3 PM-Ras-LOCKR-PL Key
FEATURES             Location/Qualifiers
     source          1..6478
                     /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      897..906
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             945..962
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             969..998
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1008..1037
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1047..1076
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2069..2086)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2075..2299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2345..2773
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2432..2451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2642..2663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2782..2802)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2787..3116
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2967..3102
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3029..3048
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3183..3977
                     /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(3237..3256)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3847..3866
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4151..4272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4188..4207)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4242..4261
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4321..4337)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4321..4337)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4334..4356)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4345..4361
                     /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(4369..4375,4376..4393,4394..4399))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4414..4435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4552..4569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4723..5311)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4803..4822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5482..6273,6274..6342))
                     /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     6105..6124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6343..6447)
                     /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 gcttgcgcca
      901 ccatgggctg catcaagagc aagaggaagg acaaagggtc tagccatcac catcatcacc
      961 attctagcga gcagaaactc atctctgaag aggatctcgg tggtagtgaa caaaagttga
     1021 tatccgagga ggatttgggt gggagcgagc agaaacttat cagtgaagaa gatttggggg
     1081 gctccatgga tcctgacgag gcaaggaaag ccatagcgag ggttaaaagg gaaagcaaac
     1141 gaatagtaga agacgcggaa aggcttagtc gagaagcagc ggcggcaagt gagaaatcct
     1201 cccgagaggc ggagaggctc tcccgggagg ccgctgctgc gtccgagaaa atttcccggg
     1261 agggcggtag tggtggaacc accgggagcg gtggtggttc cggaggacag ctggatgggg
     1321 gttccgtagc tgtgttgccg gttgtcgaca gtacaaacca gtaccttctg gatcggatcg
     1381 gtgagctgaa gtccggcgac gcctgcatag cggagtatca gcaagcgggg aggggtagca
     1441 gagggagaaa gtggttctcc ccttttgggg ccaacctcta cctttcaatg ttttggcgac
     1501 tcaaaagggg acccgcagca atcggactgg gcccggtgat tggcatcgtg atggccgagg
     1561 cactgcggaa attgggggcg gacaaggtgc gcgtaaagtg gccgaacgac ttgtatctcc
     1621 aagacagaaa actcgctggt atcctcgttg aactggcagg tatcacagga gacgcagcac
     1681 aaatcgtaat cggagctgga atcaatgtag caatgagacg cgtggaagag agtgttgtca
     1741 atcaaggttg gattactctg caggaagcag gtatcaatct cgatcggaac acgctggccg
     1801 cgactctcat tagagagctg cgagcggcac tggagttgtt cgaacaagag ggacttgcgc
     1861 cctaccttcc ccgatgggag aaactggata acttcataaa taggcccgtt aagttgataa
     1921 ttggcgataa agagattttc ggaattagta ggggtatcga caaacaaggt gccttgctcc
     1981 ttgagcaaga cggcgtgata aagccctgga tgggtggtga aatcagtctc cgaagtgcag
     2041 agaaatagga attctgcaga tatccatccc tcgactgtgc cttctagttg ccagccatct
     2101 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2161 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2221 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2281 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2341 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2401 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2461 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2521 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2581 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2641 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2701 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2761 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2821 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2881 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2941 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3001 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3061 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3121 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     3181 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3241 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3301 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3361 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3421 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3481 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3541 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3601 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3661 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3721 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3781 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3841 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3901 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3961 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     4021 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4081 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4141 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4201 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4261 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4321 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4381 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4441 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4501 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4561 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4621 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4681 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4741 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4801 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4861 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4921 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4981 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5041 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5101 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5161 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5221 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5281 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5341 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5401 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5461 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5521 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5581 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5641 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5701 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5761 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5821 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5881 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5941 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6001 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6061 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6121 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6181 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6241 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6301 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6361 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6421 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
