LOCUS       sequence-178961-        5420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A control of MCS+ that associated with cell plasma membrane by two 
            hydrophobic interaction at N and C terminus of the FRET pair..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5420)
  AUTHORS   Ma Y, Yamamoto Y, Nicovich PR, Goyette J, Rossy J, Gooding JJ, Gaus 
            K
  TITLE     A FRET sensor enables quantitative measurements of membrane charges 
            in live cells.
  JOURNAL   Nat Biotechnol. 2017 Mar 13. doi: 10.1038/nbt.3828.
  PUBMED    28288102
REFERENCE   2  (bases 1 to 5420)
  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. 2017 Mar 13. doi: 10.1038/nbt.3828."
COMMENT     Sequence Label: MCS Hras FI
FEATURES             Location/Qualifiers
     source          1..5420
                     /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"
     CDS             join(683..685,686..688,689..1201)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(728..749)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(989..1008)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1336..1357
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1497..1515)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1765..1784
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2208..2329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2245..2264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2299..2318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2336..2791)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2473..2494)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2685..2704
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2818..2922
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2924..3281
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2924..3115
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2947..2967)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3132..3267
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3316..4110
                     /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             3316..4110
                     /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(3370..3389)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3980..3999
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4298..4317)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4342..4389
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4718..5306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5207..5226
                     /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 gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attcatgggc tgtgtctgca gctcaaaccc
      661 tgaaggagga ggaggggatc ctatggtgag caagggcgag gagctgttca ccggggtggt
      721 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
      781 gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa
      841 gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc agtgcttcgc
      901 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
      961 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1021 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1081 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1141 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     1201 ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1261 cgtgctgctg cccgacaacc actacctgag ctaccagtcc aaactgagca aagaccccaa
     1321 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgcctccg gagataacat
     1381 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1441 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1501 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1561 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1621 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1681 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1741 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     1801 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     1861 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     1921 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     1981 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     2041 catgttcggc attggctgca tgagctgcaa gtgtgtgctg tcctgagcgg ccgcgactct
     2101 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     2161 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     2221 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2281 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     2341 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2401 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2461 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2521 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2581 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2641 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2701 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2761 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2821 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2881 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2941 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3001 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3061 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3121 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3181 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3241 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     3301 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3361 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3421 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3481 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3541 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3601 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3661 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3721 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3781 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3841 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3901 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3961 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4021 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4081 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4141 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4201 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4261 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     4321 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     4381 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     4441 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     4501 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     4561 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5401 accgtattac cgccatgcat
//
