LOCUS       sequence-428970-        7094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ras activity dependent proximity labeler, Cage only, untargeted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7094)
  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 7094)
  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 Cage
FEATURES             Location/Qualifiers
     source          1..7094
                     /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      926..935
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             944..961
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             968..991
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1001..1024
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1034..1057
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2685..2702)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2691..2915
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2961..3389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3048..3067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3258..3279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3398..3418)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3403..3732
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3583..3718
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3645..3664
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3799..4593
                     /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(3853..3872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4463..4482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4767..4888
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4804..4823)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4858..4877
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4937..4953)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4937..4953)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4950..4972)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4961..4977
                     /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(4985..4991,4992..5009,5010..5015))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5030..5051
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5168..5185)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5339..5927)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5419..5438)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6098..6889,6890..6958))
                     /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     6721..6740
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6959..7063)
                     /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 tcactatagg gagacccaag cttgcgccac catggggtct agccatcacc atcatcacca
      961 ttctagcgat tataaagacg atgatgacaa gggaggtagt gattacaagg atgatgacga
     1021 taaaggaggt tccgattaca aagatgatga cgacaagggc gggtctatga aggacaatac
     1081 cgtgccactc aaattgatcg cccttctcgc aaatggcgaa ttccattctg gcgaacaatt
     1141 gggggagact ttgggtatgt cacgagccgc gatcaacaag cacatacaaa cactcagaga
     1201 ttggggtgtg gatgtattta cggtcccggg aaaagggtat tccctcccgg aaccgatccc
     1261 tctcctgaat gcaaaacaaa tactcggagg cagcggggga actacagggt caggcggcgg
     1321 gtcaggatca aaggaagcag cgaagaaact ccaggacctg aatatagagc ttgcgcggaa
     1381 actcctcgaa gcgagcacga agcttcaaag acttaacata aggctggcag aagctctctt
     1441 ggaagccatc gctagattgc aggagctgaa tctggagctc gtatacctgg ctgttgagct
     1501 gacagaccct aaacggatac gggatgagat caaggaagtc aaagataaga gcaaggaaat
     1561 tattcgccgc gctgaaaaag aaatagatga tgctgccaag gagtccaaaa aaattcttga
     1621 ggaagcacga aaagctattc gagacgccgc cgaggagagt aggaaaattc tcgaggaagg
     1681 tagcgggagc gggtcagatg ccctggatga gttgcaaaag ttgaatcttg aacttgcgaa
     1741 gcttctcctt aaagcaattg ctgaaactca agacttgaat cttagagctg caaaggcctt
     1801 tctcgaagcg gccgcaaaat tgcaggaact taatattagg gctgtggaac ttctcgtaaa
     1861 gctcaccgac ccggcaacca tacgccgggc tctggagcat gctaagagac ggtcaaagga
     1921 gataatcgac gaagcggaaa gggctattcg ggcagctaag cgcgagagcg agcgaattat
     1981 tgaagaagcg cggcgactca tagagaaggc caaggaggag agtgaaagga taattcgaga
     2041 aggttccggc agtggtgatc ccgatatcaa aaaattgcag gatctcaaca ttgaactggc
     2101 aagagagctg ctccgggctc atgcacagct tcagcgactt aatttggagt tgcttcgcga
     2161 actgcttcga gctttggctc aattgcaaga gcttaacctc gacctcttga ggttggcttc
     2221 cgaattgact gacccagacg aggcgaggaa agcgatcgcc gttacaggct accggctctt
     2281 tgaagaaata ctggacgccg agcgattgag tcgggaggcc gcagcagcat ctgaaaagat
     2341 cagccgggag gctgaacgcc ttatacgaga agctgctgca accataagag tcttcctccc
     2401 gaacaaacag agaacagtcg taaatgtccg aaatgggatg tctctgcatg actgcttgat
     2461 gaaagccctc aaggtacggg gccttcagcc agagtgctgc gcggttttcc gcctcctgca
     2521 tgaacataaa ggtaagaagg cgcgactgga ctggaatacg gatgccgcct ctctcattgg
     2581 tgaggaattg caagttgact ttttgtgaga attctgcaga tatccatcac actggcggcc
     2641 gctcgagcat gcatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     2701 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2761 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2821 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2881 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2941 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3001 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3061 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3121 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3181 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3241 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3301 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3361 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3421 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3481 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3541 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3601 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3661 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3721 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3781 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3841 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3901 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3961 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4021 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4081 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4141 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4201 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4261 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4321 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4381 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4441 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4501 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4561 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4621 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4681 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4741 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4801 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4861 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4921 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4981 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5041 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5101 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5161 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5221 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5281 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5341 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5401 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5461 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5521 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5581 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5641 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5701 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5761 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5821 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5881 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5941 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6001 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6061 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6121 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6181 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6241 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6301 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6361 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6421 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6481 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6541 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6601 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6661 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6721 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6781 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6841 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6901 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6961 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7021 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7081 tgccacctga cgtc
//
