LOCUS       sequence-428968-        6520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ras activity dependent proximity labeler, Key only, untargeted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6520)
  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 6520)
  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 Ras-LOCKR-PL Key
FEATURES             Location/Qualifiers
     source          1..6520
                     /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      919..928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             937..954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             961..990
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1000..1029
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1039..1068
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2111..2128)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2117..2341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2387..2815
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2474..2493)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2684..2705
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2824..2844)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2829..3158
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3009..3144
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3071..3090
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3225..4019
                     /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(3279..3298)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3889..3908
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4193..4314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4230..4249)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4284..4303
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4363..4379)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4363..4379)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4376..4398)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4387..4403
                     /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(4411..4417,4418..4435,4436..4441))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4456..4477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4594..4611)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4765..5353)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4845..4864)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5524..6315,6316..6384))
                     /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     6147..6166
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6385..6489)
                     /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 aagcttgcgc caccatgggg tctagccatc accatcatca ccattctagc
      961 gagcagaaac tcatctctga agaggatctc ggtggtagtg aacaaaagtt gatatccgag
     1021 gaggatttgg gtgggagcga gcagaaactt atcagtgaag aagatttggg gggctccatg
     1081 gatcctgacg aggcaaggaa agccatagcg agggttaaaa gggaaagcaa acgaatagta
     1141 gaagacgcgg aaaggcttag tcgagaagca gcggcggcaa gtgagaaatc ctcccgagag
     1201 gcggagaggc tctcccggga ggccgctgct gcgtccgaga aaatttcccg ggagggcggt
     1261 agtggtggaa ccaccgggag cggtggtggt tccggaggac agctggatgg gggttccgta
     1321 gctgtgttgc cggttgtcga cagtacaaac cagtaccttc tggatcggat cggtgagctg
     1381 aagtccggcg acgcctgcat agcggagtat cagcaagcgg ggaggggtag cagagggaga
     1441 aagtggttct ccccttttgg ggccaacctc tacctttcaa tgttttggcg actcaaaagg
     1501 ggacccgcag caatcggact gggcccggtg attggcatcg tgatggccga ggcactgcgg
     1561 aaattggggg cggacaaggt gcgcgtaaag tggccgaacg acttgtatct ccaagacaga
     1621 aaactcgctg gtatcctcgt tgaactggca ggtatcacag gagacgcagc acaaatcgta
     1681 atcggagctg gaatcaatgt agcaatgaga cgcgtggaag agagtgttgt caatcaaggt
     1741 tggattactc tgcaggaagc aggtatcaat ctcgatcgga acacgctggc cgcgactctc
     1801 attagagagc tgcgagcggc actggagttg ttcgaacaag agggacttgc gccctacctt
     1861 ccccgatggg agaaactgga taacttcata aataggcccg ttaagttgat aattggcgat
     1921 aaagagattt tcggaattag taggggtatc gacaaacaag gtgccttgct ccttgagcaa
     1981 gacggcgtga taaagccctg gatgggtggt gaaatcagtc tccgaagtgc agagaaatag
     2041 gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc
     2101 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2161 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2221 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2281 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2341 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     2401 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2461 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2521 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2581 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2641 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2701 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2761 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     2821 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2881 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2941 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3001 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3061 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3121 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3181 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3241 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3301 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3361 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3421 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3481 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3541 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3601 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3661 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3721 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3781 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3841 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3901 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3961 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4021 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4081 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4141 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4201 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4261 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4321 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4381 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4441 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4501 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4561 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4621 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4681 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4741 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4801 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4861 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4921 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4981 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5041 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5101 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5161 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5221 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5281 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5341 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5401 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5461 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5521 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5581 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5641 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5701 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5761 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5821 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5881 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5941 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6001 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6061 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6121 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6181 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6241 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6301 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6361 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6421 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6481 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
