LOCUS       sequence-443640-        4807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing YFP(Venus) followed by the last 25 amino acids of
            human KRas4b including the polybasic regions and CAAX prenylation 
            box.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  AUTHORS   Luebbers A, Janicot R, Zhao J, Philibert CE, Garcia-Marcos M
  TITLE     A sensitive biosensor of endogenous Galpha(i) activity enables the 
            accurate characterization of endogenous GPCR agonist responses.
  JOURNAL   Sci Signal. 2025 Mar 25;18(879):eadp6457. doi: 
            10.1126/scisignal.adp6457. Epub 2025 Mar 25.
  PUBMED    40132053
REFERENCE   2  (bases 1 to 4807)
  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: "Sci 
            Signal."; date: "2025-03-25"; volume: "18(879):eadp6457. doi"; 
            pages: " 10.1126/scisignal.adp6457. Epub 2025 Mar 25"
COMMENT     Sequence Label: YFP-CAAX
FEATURES             Location/Qualifiers
     source          1..4807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      94..549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(181..200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     391..412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    556..677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(567..586)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     621..640
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(877..1587,1588..1590,1591..1593))
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(919..940)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1268..1287
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1527..1548
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1590..1599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1601..1681
                     /label=MCS
                     /note="multiple cloning site"
     promoter        complement(1704..1907)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1733..1753)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1908..2211
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2386..2974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2466..2485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3303..3350
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3375..3394
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3582..4376)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3582..4376)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3693..3712)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4303..4322
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4411..4768)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4425..4560
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4577..4768
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4725..4745
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(join(4770..4807,1..67))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
       61 ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg
      121 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      181 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      241 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      301 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      361 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      421 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
      481 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
      541 ttacaattta cgccttaaga tacattgatg agtttggaca aaccacaact agaatgcagt
      601 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
      661 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
      721 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
      781 atctagagtc gcggccgctc acataattac acactttgtc tttgacttct ttttcttctt
      841 tttaccatct ttgctcatct tttctttatg ttttcgcttg tacagctcgt ccatgccgag
      901 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
      961 gtctttgctc agcttggact ggtagctcag gtagtggttg tcgggcagca gcacggggcc
     1021 gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgccgc cgtcctcgat
     1081 gttgtggcgg atcttgaagt tggccttgat gccgttcttc tgcttgtcgg cggtgatata
     1141 gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt
     1201 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc
     1261 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc
     1321 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa
     1381 gcactgcagg ccgtagccga gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     1441 ggtggtgcag atgagcttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc
     1501 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     1561 cccggtgaac agctcctcgc ccttgctcac catggtggcg accggtggat cccgggcccg
     1621 cggtaccgtc gactgcagaa ttcgaagctt gagctcgaga tctgagtccg gtagcgctag
     1681 cggatctgac ggttcactaa accagctctg cttatataga cctcccaccg tacacgccta
     1741 ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgattt
     1801 tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag
     1861 tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat ggtaatagcg
     1921 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
     1981 taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
     2041 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
     2101 tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg
     2161 gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc
     2221 atatatgggc tatgaactaa tgaccccgta attgattact attaataact aatgcatggc
     2281 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2341 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2401 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2461 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2521 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2581 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2641 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2701 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2761 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2821 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2881 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2941 gcagcagatt actcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3001 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3061 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3121 atatgagtaa cctgaggcta tggcagggcc tgccgccccg acgttggctg cgagccctgg
     3181 gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa gaaacgcggg cgtattggcc
     3241 ccaatggggt ctcggtgggg tatcgacaga gtgccagccc tgggaccgaa ccccgcgttt
     3301 atgaacaaac gacccaacac cgtgcgtttt attctgtctt tttattgccg tcatagcgcg
     3361 ggttccttcc ggtattgtct ccttccgtgt ttcagttagc ctccccctag ggtgggcgaa
     3421 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     3481 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt
     3541 gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     3601 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     3661 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     3721 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     3781 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     3841 ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     3901 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     3961 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     4021 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     4081 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     4141 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt
     4201 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     4261 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     4321 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     4381 aacgatcctc atcctgtctc ttgatcgatc tttgcaaaag cctaggcctc caaaaaagcc
     4441 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
     4501 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     4561 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     4621 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     4681 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     4741 attccacagc tggttctttc cgcctcagga ctcttccttt ttcaatatta ttgaagcatt
     4801 tatcagg
//
