LOCUS       sequence-355558-        6867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RA-Hras and Lyn-iRFP682.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6867)
  AUTHORS   Kim J , Lee S, Jung K, Oh WC, Kim N, Son S, Jo Y, Kwon HB,  Heo WD
  TITLE     Intensiometric biosensors visualize the activity of multiple small 
            GTPases in vivo.
  JOURNAL   Nat Commun. 2019 Jan 14;10(1):211. doi: 10.1038/s41467-018-08217-3.
  PUBMED    30643148
REFERENCE   2  (bases 1 to 6867)
  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 
            Commun."; date: "2019-01-14"; volume: "10(1):211. doi"; pages: " 
            10.1038/s41467-018-08217-3"
COMMENT     Sequence Label: pCMV-RA-HRas-IRES2-Lyn-iRFP682
FEATURES             Location/Qualifiers
     source          1..6867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      592..601
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(731..749)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     999..1018
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1321..1890
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     primer_bind     complement(2073..2090)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2300..2319
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2475..2484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2565..2574
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2571..3518
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 682 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP682
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGVVIAEDRCAEVESKLGLHYPASAVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPCHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    3655..3776
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3692..3711)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3746..3765
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3783..4238)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3920..3941)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4132..4151
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4265..4369
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4371..4728
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4371..4562
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4394..4414)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4579..4714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4763..5557
                     /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             4763..5557
                     /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(4817..4836)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5427..5446
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5745..5764)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5789..5836
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6165..6753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6654..6673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc ggccaccatg
      601 gtgagcaaga gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg cttggagggc
      661 tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      721 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
      781 ctgtcccccc agatcatgta cggctccaag gcgtacgtga agcaccccgc cgacgtcccc
      841 gattacatga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gcacttcgag
      901 gacggcggtc tggtgaccgt gactcaggac acctccctgc aggacggcac gctgatctac
      961 aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc
     1021 ttgggctggg attatgccac cgagcgcctg taccccgaag acggcgtgct gaagggcgag
     1081 cttctggggc gcctgaagct gaaggacggc ggcctcaacc tggtggagtt caagaccatc
     1141 tacatggcca agaagcccgt gcaactgccc ggctactact tcgtggacac caagctgggc
     1201 atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc
     1261 caccacctgg ggcatggcac cggcagcacc ggcagcggca gctccggtac cctgtacaag
     1321 atgacggaat ataagctggt ggtggtgggc gccggcggtg tgggcaagag tgcgctgacc
     1381 atccagctga tccagaacca ttttgtggac gaatacgacc ccactataga ggattcctac
     1441 cggaagcagg tggtcattga tggggagacg tgcctgttgg acatcctgga taccgccggc
     1501 caggaggagt acagcgccat gcgggaccag tacatgcgca ccggggaggg cttcctgtgt
     1561 gtgtttgcca tcaacaacac caagtctttt gaggacatcc accagtacag ggagcagatc
     1621 aaacgggtga aggactcgga tgacgtgccc atggtgctgg tggggaacaa gtgtgacctg
     1681 gctgcacgca ctgtggaatc tcggcaggct caggacctcg cccgaagcta cggcatcccc
     1741 tacatcgaga cctcggccaa gacccggcag ggagtggagg atgccttcta cacgttggtg
     1801 cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc ctgatgagag tggccccggc
     1861 tgcatgagct gcaagtgtgt gctctcctaa gcgtaccggg atccgcccct ctccctcccc
     1921 cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat
     1981 gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt
     2041 cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt
     2101 gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc
     2161 gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc
     2221 acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat
     2281 agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc
     2341 ccagaaggta ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg
     2401 tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct
     2461 ttgaaaaaca cgatgccacc atgggatgta taaaatcaaa agggaaagac agcgcgggag
     2521 cagatagtgc tggtagtgct ggtagtgctg gagatccacc ggtcgccacc atggcggaag
     2581 gatccgtcgc caggcagcct gacctcttga cctgcgacga tgagccgatc catatccccg
     2641 gtgccatcca accgcatgga ctgctgctcg ccctcgccgc cgacatgacg atcgttgccg
     2701 gcagcgacaa ccttcccgaa ctcaccggac tggcgatcgg cgccctgatc ggccgctctg
     2761 cggccgatgt cttcgactcg gagacgcaca accgtctgac gatcgccttg gccgagcccg
     2821 gggcggccgt cggagcaccg atcactgtcg gcttcacgat gcgaaaggac gcaggcttca
     2881 tcggctcctg gcatcgccat gatcagctca tcttcctcga gctcgagcct ccccagcggg
     2941 acgtcgccga gccgcaggcg ttcttccgcc gcaccaacag cgccatccgc cgcctgcagg
     3001 ccgccgaaac cttggaaagc gcctgcgccg ccgcggcgca agaggtgcgg aagattaccg
     3061 gcttcgatcg ggtgatgatc tatcgcttcg cctccgactt cagcggggtg gtgatcgcag
     3121 aggatcgatg cgccgaggtc gagtcaaaac taggcctgca ctatcctgcc tcagcggtgc
     3181 cggcgcaggc ccgtcggctc tataccatca acccggtacg gatcattccc gatatcaatt
     3241 atcggccggt gccggtcacc ccagacctca atccggtcac cgggcggccg attgatctta
     3301 gcttcgccat cctgcgcagc gtctcgccct gccatttgga gttcatgcgc aacataggca
     3361 tgcacggcac gatgtcgatc tcgattttgc gcggcgagcg actgtgggga ttgatcgttt
     3421 gccatcaccg aacgccgtac tacgtcgatc tcgatggccg ccaagcctgc gagctagtcg
     3481 cccaggttct ggcctggcag atcggcgtga tggaagagct gtacaagtaa caccggatct
     3541 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3601 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3661 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3721 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc
     3781 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3841 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3901 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3961 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4021 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4081 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4141 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4201 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4381 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4441 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4501 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4561 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4621 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4681 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4741 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4801 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4861 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4921 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4981 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5041 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5101 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5161 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5221 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5281 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5341 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5401 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5461 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5521 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5581 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5641 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5701 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5761 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5821 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5881 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5941 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     6001 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6301 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6781 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6841 gtggataacc gtattaccgc catgcat
//
