LOCUS       sequence-422203-        7088 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 7088)
  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 7088)
  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..7088
                     /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             915..932
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             939..962
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             972..995
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1005..1028
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2640..2658)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2641..2658)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2678..2695)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2684..2908
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2954..3382
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3041..3060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3251..3272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3391..3411)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3396..3726
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3577..3712
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3639..3658
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3793..4587
                     /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(3847..3866)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4457..4476
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4761..4882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4798..4817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4852..4871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4931..4947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4931..4947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4944..4966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4955..4971
                     /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(4979..4985,4986..5003,5004..5009))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5024..5045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5162..5179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5333..5921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5413..5432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6092..6883,6884..6952))
                     /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     6715..6734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6953..7057)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 ccatggggtc tagccatcac catcatcacc attctagcga ttataaagac gatgatgaca
      961 agggaggtag tgattacaag gatgatgacg ataaaggagg ttccgattac aaagatgatg
     1021 acgacaaggg cgggtctatg aaggacaata ccgtgccact caaattgatc gcccttctcg
     1081 caaatggcga attccattct ggcgaacaat tgggggagac tttgggtatg tcacgagccg
     1141 cgatcaacaa gcacatacaa acactcagag attggggtgt ggatgtattt acggtcccgg
     1201 gaaaagggta ttccctcccg gaaccgatcc ctctcctgaa tgcaaaacaa atactcggag
     1261 gcagcggggg aactacaggg tcaggcggcg ggtcaggatc aaaggaagca gcgaagaaac
     1321 tccaggacct gaatatagag cttgcgcgga aactcctcga agcgagcacg aagcttcaaa
     1381 gacttaacat aaggctggca gaagctctct tggaagccat cgctagattg caggagctga
     1441 atctggagct cgtatacctg gctgttgagc tgacagaccc taaacggata cgggatgaga
     1501 tcaaggaagt caaagataag agcaaggaaa ttattcgccg cgctgaaaaa gaaatagatg
     1561 atgctgccaa ggagtccaaa aaaattcttg aggaagcacg aaaagctatt cgagacgccg
     1621 ccgaggagag taggaaaatt ctcgaggaag gtagcgggag cgggtcagat gccctggatg
     1681 agttgcaaaa gttgaatctt gaacttgcga agcttctcct taaagcaatt gctgaaactc
     1741 aagacttgaa tcttagagct gcaaaggcct ttctcgaagc ggccgcaaaa ttgcaggaac
     1801 ttaatattag ggctgtggaa cttctcgtaa agctcaccga cccggcaacc atacgccggg
     1861 ctctggagca tgctaagaga cggtcaaagg agataatcga cgaagcggaa agggctattc
     1921 gggcagctaa gcgcgagagc gagcgaatta ttgaagaagc gcggcgactc atagagaagg
     1981 ccaaggagga gagtgaaagg ataattcgag aaggttccgg cagtggtgat cccgatatca
     2041 aaaaattgca ggatctcaac attgaactgg caagagagct gctccgggct catgcacagc
     2101 ttcagcgact taatttggag ttgcttcgcg aactgcttcg agctttggct caattgcaag
     2161 agcttaacct cgacctcttg aggttggctt ccgaattgac tgacccagac gaggcgagga
     2221 aagcgatcgc cgttacaggc taccggctct ttgaagaaat actggacgcc gagcgattga
     2281 gtcgggaggc cgcagcagca tctgaaaaga tcagccggga ggctgaacgc cttatacgag
     2341 aagctgctgc aaccataaga gtcttcctcc cgaacaaaca gagaacagtc gtaaatgtcc
     2401 gaaatgggat gtctctgcat gactgcttga tgaaagccct caaggtacgg ggccttcagc
     2461 cagagtgctg cgcggttttc cgcctcctgc atgaacataa aggtaagaag gcgcgactgg
     2521 actggaatac ggatgccgcc tctctcattg gtgaggaatt gcaagttgac tttttgtgag
     2581 aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga gggccctatt
     2641 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     2701 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2761 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2821 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2881 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2941 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3001 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3061 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta
     3121 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3181 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3241 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3301 tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt
     3361 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3421 ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3481 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3541 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3601 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3661 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3721 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3781 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3841 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3901 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3961 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4021 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4081 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4141 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4201 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4261 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4321 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4381 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4441 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4501 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4561 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4621 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4681 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4741 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4801 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4861 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4921 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4981 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5041 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5101 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5161 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5221 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5281 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5341 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5401 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5461 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5521 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5581 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5641 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5701 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5761 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5821 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5881 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5941 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6001 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6061 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6121 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6181 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6241 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6301 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6361 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6421 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6481 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6541 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6601 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6661 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6721 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6781 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6841 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6901 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6961 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7021 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7081 ctgacgtc
//
