LOCUS       sequence-363186-        7362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of FluoSTEP-RhoA biosensor; must be expressed with GFP11-tagged
            RhoA plasmid (Addgene #181860) or in GFP11-RhoA gene-edited cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7362)
  AUTHORS   Tenner B, Zhang JZ, Kwon Y, Pessino V, Feng S, Huang B, Mehta S, 
            Zhang J
  TITLE     FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized 
            signaling within endogenous microdomains.
  JOURNAL   Sci Adv. 2021 May 21;7(21). pii: 7/21/eabe4091. doi: 
            10.1126/sciadv.abe4091. Print 2021 May.
  PUBMED    34020947
REFERENCE   2  (bases 1 to 7362)
  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 Adv."; 
            date: "2021-05-21"; volume: "7(21). pii: 7/21/eabe4091. doi"; pages:
            " 10.1126/sciadv.abe4091. Print 2021 May"
COMMENT     Sequence Label: pcDNA3.1(+)-cpPKN-mRuby2-GFP1-10
FEATURES             Location/Qualifiers
     source          1..7362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     733..752
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        733..751
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1071..1781
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RITVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREVAV
                     AKFAGLGGGMDELYK"
     CDS             1797..2144
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             2148..2789
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     primer_bind     complement(2824..2841)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2830..3054
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3100..3527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3187..3206)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3396..3417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3536..3556)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3541..3870
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3721..3856
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3783..3802
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3937..4731
                     /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(3991..4010)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4601..4620
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4905..5026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4942..4961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4996..5015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5075..5091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5075..5091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5088..5110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5099..5115
                     /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(5123..5129,5130..5147,5148..5153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5168..5189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5306..5323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5557..5576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6236..7027,7028..7096))
                     /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     6859..6878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7097..7201)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7276..7295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      721 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
      781 aagcttggat gagcctgggc cccgtagagc tgctgctgcg gggctcctcg cgccgcctcg
      841 acctgctgca ccagcagctg caggagctgc acgcccacgg ttctggttcc ctgctagagc
      901 agctgggcct ggccggggca gacctggcgg cccccggggt acagcagcag ctggagctgg
      961 agcgggagcg gctgcggcgg gaaatccgca aggagctgaa gctgaaggag ggtgctgaga
     1021 acctgcggcg ggccaccact gacctgggcc gcggtggaac aggaggttca atggtgtcta
     1081 agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca
     1141 acggccacca attcaaatgc acaggtgaag gagaaggcaa tccgtacatg ggaactcaaa
     1201 ccatgaggat cacagtcatc gagggaggac ccctgccatt tgcctttgac attcttgcca
     1261 cgtcgttcat gtatggcagc cgtactttta tcaagtaccc gaaaggcatt cctgatttct
     1321 ttaaacagtc ctttcctgag ggttttactt gggaaagagt tacgagatac gaagatggtg
     1381 gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg ctgtctcgtt taccacgtcc
     1441 aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag accaagggtt
     1501 gggagcctaa tacagagatg atgtatccag cagatggtgg tctgagggga tacactcata
     1561 tggcactgaa agttgatggt ggtggccatc tgtcttgctc tttcgtaaca acttacaggt
     1621 caaaaaagac cgtcgggaac atcaagatgc ccggtatcca tgccgttgat caccgcctgg
     1681 aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca acgcgaagtc gcagttgcca
     1741 agttcgccgg gcttggtggt gggatggacg agctgtacaa gggctccgga ggaaccagtg
     1801 ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggttccg
     1861 gcagtgctgg tggtagtgct ggtggtagta ccagtgctgg tggtagtgct ggtggtagtg
     1921 ctggtggtag tgctggtggt agtgctggtg gttccggcag tgctggtggt agtgctggtg
     1981 gtagtaccag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg
     2041 ctggtggttc cggcagtgct ggtggtagtg ctggtggtag taccagtgct ggtggtagtg
     2101 ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggtaccatg tccaaaggag
     2161 aagaactgtt taccggtgtt gtgccaattt tggttgaact cgatggtgat gtcaacggac
     2221 ataagttctc agtgagaggc gaaggagaag gtgacgccac cattggaaaa ttgactctta
     2281 aattcatctg tactactggt aaacttcctg taccatggcc gactctcgta acaacgctta
     2341 cgtacggagt tcagtgcttt tcgagatacc cagaccatat gaaaagacat gactttttta
     2401 agtcggctat gcctgaaggt tacgtgcaag aaagaacaat ttcgttcaaa gatgatggaa
     2461 aatataaaac tagagcagtt gttaaatttg aaggagatac tttggttaac cgcattgaac
     2521 tgaaaggaac agattttaaa gaagatggta atattcttgg acacaaactc gaatacaatt
     2581 ttaatagtca taacgtatac atcactgctg ataagcaaaa gaacggaatt aaagcgaatt
     2641 tcacagtacg ccataatgta gaagatggca gtgttcaact tgccgaccat taccaacaaa
     2701 acacccctat tggagacggt ccggtacttc ttcctgataa tcactacctc tcaacacaaa
     2761 cagtcctgag caaagatcca aatgaaaaat agtctagagg gcccgtttaa acccgctgat
     2821 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2881 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2941 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3001 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3061 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3121 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3181 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3241 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3301 ccaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3361 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3421 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3481 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3541 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3601 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3661 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3721 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3781 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3841 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttatat atccattttc
     3901 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3961 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4021 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4081 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4141 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4201 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4261 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4321 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4381 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4441 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4501 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4561 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4621 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4681 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4741 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4801 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4861 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4921 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4981 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5041 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5101 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5161 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5221 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5281 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5881 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6121 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6181 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6541 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7081 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7141 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7201 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7261 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7321 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa at
//
