LOCUS       sequence-338269-        7346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET biosensor. To monitor Src kinase activity in mammalian cells by
            FRET.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7346)
  AUTHORS   Ouyang M, Sun J, Chien S, Wang Y
  TITLE     Determination of hierarchical relationship of Src and Rac at 
            subcellular locations with FRET biosensors.
  JOURNAL   Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14353-8. doi: 
            10.1073/pnas.0807537105. Epub 2008 Sep 17.
  PUBMED    18799748
REFERENCE   2  (bases 1 to 7346)
  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"; doi: 
            "10.1073/pnas.0807537105"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2008-09-23- 23"; volume: "105"; issue: "38"; pages: "14353-8"
COMMENT     Sequence Label: Kras-Src FRET biosensor
FEATURES             Location/Qualifiers
     source          1..7346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             688..705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             709..741
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(745..753,754..768)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(775..777,778..780,781..1293)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     primer_bind     complement(820..841)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1081..1100)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1428..1449
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1849..2559
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
                     LTAAGITEGMNELYK"
     promoter        complement(2665..2683)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2666..2683)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2703..2720)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2709..2933
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2979..3407
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3066..3085)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3276..3297
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3416..3436)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3421..3750
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3601..3736
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3663..3682
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3817..4611
                     /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(3871..3890)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4481..4500
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4785..4906
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4822..4841)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4876..4895
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4955..4971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4955..4971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4968..4990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4979..4995
                     /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(5003..5009,5010..5027,5028..5033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5048..5069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5186..5203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5357..5945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5437..5456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6116..6907,6908..6976))
                     /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     6739..6758
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6977..7081)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7156..7175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gcggccgcca ccatggtgcg gggttctcat catcatcatc atcatggtat ggctagcatg
      721 actggtggac agcaaatggg tcgggatctg tacgacgatg acgataagga tcccatggtg
      781 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      841 gtaaacggcc acaggttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      901 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      961 accaccctga cctggggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1021 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgtaccat cttcttcaag
     1081 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1141 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1201 gagtacaact acatcagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     1261 aaggcccact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1321 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1381 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1441 gagttcgtga ccgccgcccg catgcattgg tattttggga agatcactcg tcgggagtcc
     1501 gagcggctgc tgctcaaccc cgaaaacccc cggggaacct tcttggtccg ggagagcgag
     1561 acgacaaaag gtgcctattg cctctccgtt tctgactttg acaacgccaa ggggctcaat
     1621 gtgaagcact acaagatccg caagctggac agcggcggct tctacatcac ctcacgcaca
     1681 cagttcagca gcctgcagca gctggtggcc tactactcca aacatgctga tggcttgtgc
     1741 caccgcctga ctaacgtctg tgggtctaca tctggatctg ggaagccggg ttctggtgag
     1801 ggttcttgga tggaggacta tgactacgtc cacctacagg gggagctctc taaaggtgaa
     1861 gaattattca ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac
     1921 aaattttctg tctccggtga aggtgaaggt gatgctacgt acggtaaatt gaccttaaaa
     1981 ttactctgta ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaggt
     2041 tatggtgttc aatgttttgc tagataccca gatcatatga aacaacatga ctttttcaag
     2101 tctgccatgc cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac
     2161 tacaagacca gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta
     2221 aaaggtattg attttaaaga agatggtaac attttaggtc acaaattgga atacaactat
     2281 aactctcaca atgtttacat cactgctgac aaacaaaaga atggtatcaa agctaacttc
     2341 aaaattagac acaacattga agatggtggt gttcaattag ctgaccatta tcaacaaaat
     2401 actccaattg gtgatggtcc agtcttgtta ccagacaacc attacttatc ctatcaatct
     2461 gccttattca aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttttgact
     2521 gctgctggta ttaccgaggg tatgaatgaa ttgtacaaaa agaagaagaa gaagaagagc
     2581 aagaccaagt gcgtgatcat gtaagaattc tgcagatatc catcacactg gcggccgctc
     2641 gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2701 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2761 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2821 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2881 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2941 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3001 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3061 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3121 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3181 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3241 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3301 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3361 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3421 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3481 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3541 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3601 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3661 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3721 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3781 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3841 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3901 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3961 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4021 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4081 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4141 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4201 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4261 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4321 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4381 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4441 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4501 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4561 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4621 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4681 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4741 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4801 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4861 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4921 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4981 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5041 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5101 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5161 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5221 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5281 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5341 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5401 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5461 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5521 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5581 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5641 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5701 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5761 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5821 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5881 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5941 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6001 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6061 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6121 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6181 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6241 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6301 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6361 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6421 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6481 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6541 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6601 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6661 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6721 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6781 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6841 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6901 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6961 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7021 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7081 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7141 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7201 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7261 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     7321 cgatgtacgg gccagatata cgcgtt
//
