LOCUS       sequence-428807-        6535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ras activity dependent proximity labeler, Key only, mitochondria 
            localized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6535)
  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 6535)
  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 Mito-Ras-LOCKR-PL Key
FEATURES             Location/Qualifiers
     source          1..6535
                     /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             1002..1019
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1026..1055
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1065..1094
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1104..1133
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2126..2143)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2132..2356
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2402..2830
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2489..2508)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2699..2720
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2839..2859)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2844..3173
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3024..3159
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3086..3105
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3240..4034
                     /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(3294..3313)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3904..3923
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4208..4329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4245..4264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4299..4318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4378..4394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4378..4394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4391..4413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4402..4418
                     /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(4426..4432,4433..4450,4451..4456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4471..4492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4609..4626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4780..5368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4860..4879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5539..6330,6331..6399))
                     /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     6162..6181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6400..6504)
                     /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 ccatggcaat ccaattgcgt tcgctcttcc ccttggcgtt gcccggaatg ctggccctcc
      961 ttggctggtg gtggtttttc tctcgtaaaa aagggtctag ccatcaccat catcaccatt
     1021 ctagcgagca gaaactcatc tctgaagagg atctcggtgg tagtgaacaa aagttgatat
     1081 ccgaggagga tttgggtggg agcgagcaga aacttatcag tgaagaagat ttggggggct
     1141 ccatggatcc tgacgaggca aggaaagcca tagcgagggt taaaagggaa agcaaacgaa
     1201 tagtagaaga cgcggaaagg cttagtcgag aagcagcggc ggcaagtgag aaatcctccc
     1261 gagaggcgga gaggctctcc cgggaggccg ctgctgcgtc cgagaaaatt tcccgggagg
     1321 gcggtagtgg tggaaccacc gggagcggtg gtggttccgg aggacagctg gatgggggtt
     1381 ccgtagctgt gttgccggtt gtcgacagta caaaccagta ccttctggat cggatcggtg
     1441 agctgaagtc cggcgacgcc tgcatagcgg agtatcagca agcggggagg ggtagcagag
     1501 ggagaaagtg gttctcccct tttggggcca acctctacct ttcaatgttt tggcgactca
     1561 aaaggggacc cgcagcaatc ggactgggcc cggtgattgg catcgtgatg gccgaggcac
     1621 tgcggaaatt gggggcggac aaggtgcgcg taaagtggcc gaacgacttg tatctccaag
     1681 acagaaaact cgctggtatc ctcgttgaac tggcaggtat cacaggagac gcagcacaaa
     1741 tcgtaatcgg agctggaatc aatgtagcaa tgagacgcgt ggaagagagt gttgtcaatc
     1801 aaggttggat tactctgcag gaagcaggta tcaatctcga tcggaacacg ctggccgcga
     1861 ctctcattag agagctgcga gcggcactgg agttgttcga acaagaggga cttgcgccct
     1921 accttccccg atgggagaaa ctggataact tcataaatag gcccgttaag ttgataattg
     1981 gcgataaaga gattttcgga attagtaggg gtatcgacaa acaaggtgcc ttgctccttg
     2041 agcaagacgg cgtgataaag ccctggatgg gtggtgaaat cagtctccga agtgcagaga
     2101 aataggaatt ctgcagatat ccatccctcg actgtgcctt ctagttgcca gccatctgtt
     2161 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2221 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2281 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2341 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2401 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2461 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2521 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2581 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2641 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2701 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2761 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2821 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2881 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2941 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3001 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3061 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3121 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3181 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3241 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3301 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3361 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3421 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3481 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3541 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3601 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3661 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3721 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     3781 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3841 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3901 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3961 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4021 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4081 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4141 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4201 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4261 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4321 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4381 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4441 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4501 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4561 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4621 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4681 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4801 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4861 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4921 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4981 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5041 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5101 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5161 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5221 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5281 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5341 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5401 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5521 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5581 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5641 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5701 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5761 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5821 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5881 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5941 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6001 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6061 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6121 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6181 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6241 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6301 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6361 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6421 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6481 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
