LOCUS       sequence-428812-        6544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ras activity dependent proximity labeler, Key only, Golgi localized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6544)
  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 6544)
  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 Golgi-Ras-LOCKR-PL Key
FEATURES             Location/Qualifiers
     source          1..6544
                     /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             1011..1028
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1035..1064
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1074..1103
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1113..1142
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2135..2152)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2141..2365
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2411..2839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2498..2517)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2708..2729
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2848..2868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2853..3182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3033..3168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3095..3114
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3249..4043
                     /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(3303..3322)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3913..3932
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4217..4338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4254..4273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4308..4327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4387..4403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4387..4403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4400..4422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4411..4427
                     /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(4435..4441,4442..4459,4460..4465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4480..4501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4618..4635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4789..5377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4869..4888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5548..6339,6340..6408))
                     /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     6171..6190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6409..6513)
                     /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 ccatgggtaa cttgaaatca gttgcgcagg agccaggtcc accatgtggg ctggggctgg
      961 gtctcgggct tggactctgc ggtaagcagg gccccgcgac tgggtctagc catcaccatc
     1021 atcaccattc tagcgagcag aaactcatct ctgaagagga tctcggtggt agtgaacaaa
     1081 agttgatatc cgaggaggat ttgggtggga gcgagcagaa acttatcagt gaagaagatt
     1141 tggggggctc catggatcct gacgaggcaa ggaaagccat agcgagggtt aaaagggaaa
     1201 gcaaacgaat agtagaagac gcggaaaggc ttagtcgaga agcagcggcg gcaagtgaga
     1261 aatcctcccg agaggcggag aggctctccc gggaggccgc tgctgcgtcc gagaaaattt
     1321 cccgggaggg cggtagtggt ggaaccaccg ggagcggtgg tggttccgga ggacagctgg
     1381 atgggggttc cgtagctgtg ttgccggttg tcgacagtac aaaccagtac cttctggatc
     1441 ggatcggtga gctgaagtcc ggcgacgcct gcatagcgga gtatcagcaa gcggggaggg
     1501 gtagcagagg gagaaagtgg ttctcccctt ttggggccaa cctctacctt tcaatgtttt
     1561 ggcgactcaa aaggggaccc gcagcaatcg gactgggccc ggtgattggc atcgtgatgg
     1621 ccgaggcact gcggaaattg ggggcggaca aggtgcgcgt aaagtggccg aacgacttgt
     1681 atctccaaga cagaaaactc gctggtatcc tcgttgaact ggcaggtatc acaggagacg
     1741 cagcacaaat cgtaatcgga gctggaatca atgtagcaat gagacgcgtg gaagagagtg
     1801 ttgtcaatca aggttggatt actctgcagg aagcaggtat caatctcgat cggaacacgc
     1861 tggccgcgac tctcattaga gagctgcgag cggcactgga gttgttcgaa caagagggac
     1921 ttgcgcccta ccttccccga tgggagaaac tggataactt cataaatagg cccgttaagt
     1981 tgataattgg cgataaagag attttcggaa ttagtagggg tatcgacaaa caaggtgcct
     2041 tgctccttga gcaagacggc gtgataaagc cctggatggg tggtgaaatc agtctccgaa
     2101 gtgcagagaa ataggaattc tgcagatatc catccctcga ctgtgccttc tagttgccag
     2161 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2221 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2281 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2341 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2401 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2461 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2521 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2581 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2641 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2701 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2761 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2821 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2881 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2941 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3001 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3061 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3121 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3181 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3241 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3301 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3361 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3421 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3481 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3541 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3601 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3661 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3721 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     3781 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     3841 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     3901 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     3961 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4021 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4081 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4141 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4201 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4261 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4321 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4381 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4441 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4501 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4621 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4681 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4741 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4801 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4861 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4921 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4981 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5041 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5101 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5161 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5221 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5281 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5341 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5401 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5461 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5521 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5581 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5641 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5701 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5761 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5821 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5881 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5941 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6001 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6061 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6121 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6181 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6241 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6301 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6361 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6421 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6481 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6541 cgtc
//
